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THESIS

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This is to certify that the

thesis entitled

Utilization Of Okara To Produce

Extruded Cereal Products

presented by

Vincent E.A. Rinaldi r

has been accepted towards fulfillment
of the requirements for

Master OF Science degree inlooLScience

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Utilization Of Okara To Produce Extruded Cereal Products

By

Vincent E.A. Rinaldi

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Food Science and Human Nutrition

1 998

ABSTRACT
THE UTILIZATION or OKARA To PRODUCE EXTRUDED CEREAL
PRODUCTS
By

\fincent E.A. Rinaldi

Okara is the residue that remains after soymilk and/or tofu production. In
North America, okara is either used as animal feed, fertilizer, or landfill. Okara
has been shown to have promising nutritional attributes, however, a high
moisture content and susceptibility to putrefaction have limited its use. The
purpose of this study was to use wet okara to produce and enrich extruded cereal
products, and to study the effects on the dietary fiber and isoflavone contents due
to extrusion. Wet okara was combined with soft wheat flour to produce two
formulations and extruded using four screw configuration/temperature profile
conditions. Various physicochemical properties, dietary fiber by enzymatic-
gravimetric method, and isoflavone content by high performance liquid
chromatography were analyzed. Physicochemical analyses showed varied
results. Combining okara with soft wheat flour resulted in increased protein,
dietary fiber, and isoflavone contents compared to soft wheat flour alone.
Extrusion of the formulations resulted in decreased insoluble fiber and increased
soluble fiber content of extrudates. Extrusion decreased the total detectable

isoflavones and altered the distribution of the six detected isoflavones.

ACKNOWLEDGMENTS

I would like to express my sincere gratitude and thanks to Dr. P.K.W. Ng
for serving as my advisor, for his scientific insight, and above all for being my
friend. Many thanks go to Dr. M.R. Bennink and Dr. J.F. Steffe for serving as
members of my graduate guidance committee, for their technical advice, and their
support.

I greatly appreciate the financial support received from Dr. P.K.W. Ng and
the Food Science and Human Nutrition Department of Michigan State University;
without an assistantship it would have been difficult to have completed my
studies.

I would like to express my thanks to my lab mates: M. Accerbi, A.
Anderson, P. Cesar, L. Lee, S. Lee. and R. Redaelli, for their friendship and
support. I would like to express special thanks to my good friends Sam and Amy
Hazen who were always there when I needed them. I would also like to thank my
good friend and riding partner Jeff Novotony who always kept me going when I
wanted to quit.

Last but not least I would like to express my deepest love and gratitude to
the most important people in my life: my significant other, Ellen Hudson, whose
love and support is more than I can repay; my parents, VIncenzo and Della, and
my sister, Mary Jo, who were always understanding, loving, and encouraging.
Without these four people I would not have been able to accomplish this

monumental task.

iii

This thesis is dedicated to my parents who have always sacrificed in order

to provide my sister and me with the advantages in life that they never had.

iv

TABLE OF CONTENTS

Chapter Page
LIST OF TABLES .............................................................................................. ix
LIST OF FIGURES ............................................................................................ xi
I. INTRODUCTION ............................................................................................. 1
ll. LITERATURE REVIEW .................................................................................. 4
2.1 Soybean Seed Structure and Composition .............................................. 4
2.2 Soymilk and Tofu Production .................................................................. 5
2.3 Okara and its Composition ..................................................................... 7
2.4 Utilization of Okara ................................................................................. 7
2.5 Dietary Fiber ........................................................................................... 8
2.5.1 Health Benefits of Dietary Fiber ...................................................... 9
2.5.2 Effects of Extrusion on Dietary Fiber ............................................... 9
2.5.3 Determination of Dietary Fiber ....................................................... 10
2.6 lsoflavones ............................................................................................ 11
2.6.1 Health Benefits of Isoflavones ....................................................... 13
2.6.2 Effects of Processing on Isoflavones ............................................. 13
2.6.3 lsoflavone Determination ............................................................... 14
2.7 Soy Proteins .......................................................................................... 15
2.7.1 Amino Acid Composition of Proteins in Soybeans and Okara ........ 16
2.7.2 Processing Effects on Amino Acids ............................................... 16
2.8 Extrusion ............................................................................................... 18

2.8.1 Shear and Heat ............................................................................. 20

2.8.2 Determination of Physical Characteristics of Extruded Products....22

lIl. MATERIALS AND METHODS ...................................................................... 24
3.1 Materials ................................................................................................ 24
3.2 Methods ................................................................................................. 24

3.2.1 Preparation of Formulations ........................................................... 24
3.2.2 Extrusion Equipment ...................................................................... 26
3.2.3 Extrusion Parameters ..................................................................... 26
3.2.4 Extrusion Process .......................................................................... 28
3.2.5 Proximate Analysis of Samples ...................................................... 30
3.2.6 Radial Expansion Ratio, Bulk Density, and Breaking Strength ....... 31
3.2.7 Water Absorption Index and Water Solubility Index ....................... 32
3.2.8 Analysis of Pasting Properties ....................................................... 32
3.2.9 Specific Mechanical Energy ........................................................... 33
3.2.10 Dietary Fiber Analysis .................................................................. 35
3.2.11 Analysis of Isoflavones ................................................................ 36
3.3 Statistical Analysis ................................................................................. 39

IV. RESULTS AND DISCUSSION ..................................................................... 40
4.1 Proximate Analysis of Non-Extruded and Extruded Samples ................. 40
4.2 Physicochemical Analysis of Non-Extruded and Extruded Samples ....... 42

4.2.1 Radial Expansion Ratio, Bulk Density, and Breaking Strength ....... 42
4.2.2 Water Absorption Index and Water Solubility Index ....................... 44
4.2.3 Pasting Properties ......................................................................... 47

vi

4.2.4 Specific Mechanical Energy ........................................................... 54

4.3 Dietary Fiber Contents in Non-Extruded and Extruded Samples ............ 56
4.4 Isoflavone Contents in Non-Extruded and Extruded Samples ................ 62
V. SUMMARY AND CONCLUSIONS ................................................................ 70
VI. SUGGESTIONS FOR FUTURE RESEARCH .............................................. 72
APPENDICES ................................................................................................... 75

APPENDIX l: Flour Characteristics Determined by Farinograph Constant
Weight Method ........................................................................ 75

APPENDIX II: Raw Data for Moisture Analysis of the Non-Extruded and
Extruded Samples ................................................................. 76

APPENDIX III: Raw Data for Protein Analysis of the Non-Extruded and
Extruded Samples ................................................................ 77

APPENDIX IV: Raw Data for Ash Analysis of the Non-Extruded and
Extruded Samples ................................................................ 78

APPENDIX V: Raw Data for Fat Analysis of the Non-Extruded and
Extruded Samples ................................................................. 79

APPENDIX VI: Raw Data for Water Absorption Index Analysis of the Non-
Extruded and Extruded Samples ......................................... 80

APPENDIX VII: Raw Data for Water Solubility Index Analysis of the Non-
Extruded and Extruded Samples ........................................ 81

APPENDIX VIII: Raw Data for Radial Expansion Ratio Analysis of the
Extruded Samples .............................................................. 82

APPENDIX IX: Raw Data for Bulk Density Analysis of the Extruded
Samples ............................................................................... 83

APPENDIX X: Raw Data for Breaking Strength Analysis of the Extruded
Samples ................................................................................ 84

APPENDIX XI: Raw Data for VIscosity Properties of the Non-Extruded
and Extruded Samples ......................................................... 85

vii

APPENDIX XII: Raw Data for Screw Speed, Torque, Feed Rate, and
Specific Mechanical Energy of the Extruded Samples .......... 86

APPENDIX XIII: Raw Data for Insoluble, Soluble, and Total Dietary Fiber
Analysis of the Non-Extruded and Extruded Samples ......... 87

APPENDIX XIV: Raw Data for lsoflavone Analysis of the Non-Extruded
and Extruded Samples ........................................................ 88

BIBLIOGRAPHY ................................................................................................ 89

viii

LIST OF TABLES

Table Page
2.1 Average Amino Acid Composition ............................................................... 17
3.1 Proportions by Weight of Okara and Soft Wheat Flour Used for

Formulations A and B ................................................................................. 25
3.2 Screw Elements Used for Low Shear and High Shear Configurations ......... 27
3.3 Temperature of the Five Heating Zones Used for the

Low Temperature (LT) and High Temperature (HT) Profiles ...................... 29
3.4 Pasting Profile Used for RVA Analysis ........................................................ 34
3.5 Linear Gradient Program Used for HPLC Analysis of Isoflavones ............... 38
4.1 Proximate Analysis Data for Non-Extruded and Extruded Samples ............. 41

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

Radial Expansion Ratio, Bulk Density, and Breaking
Strength for Extruded Samples ................................................................... 43

Water Absorption Index (WAI) and Water Solubility Index (WSI)
for Non-Extruded and Extruded Products ................................................... 45

Rapid VIsco-Analyser Data for Non-Extruded Formulations A and B,
Non—Extruded Flour, and Extruded FlourNVater at the HSIHT Condition ..... 48

Rapid VIsco-Analyser Data for Extruded Formulations A and B Samples....52
Torque, Feed Rate, and Specific Mechanical Energy (SME)

Data for Extruded Formulations A and B, and Extruded FlourNVater

at the HSIHT Condition .............................................................................. 55
Insoluble, Soluble, and Total Dietary Fiber Content of Non-Extruded

Okara, Formulations A and B, and Comparison Between Non-Extruded

Flour and Extruded FlourNVater at the HSIHT Condition ............................. 57

Insoluble, Soluble, and Total Dietary Fiber Contents for
Non-Extruded and Extruded Formulations A and B ..................................... 59

Individual and Total Detected Isoflavone Contents of
Non-Extruded Samples ............................................................................... 64

ix

4.10 Individual and Total Detected lsoflavone Contents
for Non-Extruded and Extruded Formulation A Samples ............................ 66

4.11 Individual and Total Detected Isoflavone Contents
for Non-Extruded and Extruded Formulation B Samples ............................ 67

LIST OF FIGURES

Figure Page
2.1 Structures of Isoflavone Isomers in Soybeans ............................................. 12

4.1 Rapid VIscograms for Non-Extruded Flour Sample and Extruded
FlourNVater Sample at the High Shear/High Temperature Condition ........... 50

4.2 HPLC Chromatogram for Okara Depicting Detected Isoflavones
in Order of Elution ....................................................................................... 63

xi

CHAPTER I

INTRODUCTION

Soybeans are an ancient crop that reportedly originated in Eastern Asia and
only recently made their way to the United States as a major food crop (Waggle and
Kolar 1979). In 1995, the United States produced approximately 58.56 million metric
tons of soybeans (ASA 1995). The US. is the single largest producer of soybeans
followed by Brazil, Argentina, and China (Kluis 1997).

Soybeans provide a major nutritional protein source for many people. This is
of particular concern for the immediate future since it is estimated that by the year
2000 the world population will exceed six billion (Salunkhe and Kadam 1984).
Soybeans have many distinctive functional and nutritional properties (Lo 1989). The
most notable attributes of soybeans are their oil and protein contents, however they
are also a good source of fiber and isoflavones. The soybean represents an
inexpensive source of high quality protein, which may be particularly important for
developing countries (Messina 1995). Isoflavones are phytochemicals and have
been speculated to have anticarcinogenic effects. If this proves true, this places
particular emphasis on soy because isoflavones have limited distribution in nature
(Messina 1995). Evidence has also mounted that increasing consumption of a high
quality source of dietary fiber not only can improve digestion and the elimination
process, but also can aid in the treatment and prevention of diet-related disorders

such as obesity, cardiovascular disease, and diabetes (Lo 1989).

Throughout their existence, soybeans have been utilized to make many food
products. Soyfoods are typically divided into two categories: non-fermented (soy
flour, soymilk, tofu, and okara) and fermented (tempeh. misc, and soy sauce)
(Golbitz 1995). Two popular products in the US. are tofu and soymilk. The
production of each of these results in a byproduct called okara. In the US, okara is
typically discarded, but may also be used as animal feed or fertilizer. In Japan,
however, it is used as a food source (e.g., okara tempeh). In any country,
discarding okara as waste is potentially an environmental problem because okara is
highly susceptible to putrefaction. Okara has been shown to have nutritional
attributes and finding convenient ways to incorporate it into food products are
needed. This would also help to eliminate possible pollution problems and to add
economic value to okara. Extrusion could provide a convenient and low cost way to
incorporate the okara into food products.

Okara reportedly contains about 20% vegetable protein (on a dry basis) and
large amounts of dietary fiber (Watanabe and Kishi 1984). It has been shown that
soybeans and their products, tofu and soymilk, contain isoflavones (Dwyer et al
1994), as does their byproduct okara (Wang and Murphy 1996). In light of the
attributes of okara. it is a suitable candidate for nutritional enrichment for cereal-
based products. Okara could increase the protein, dietary fiber, and isoflavone
contents of these products.

The objectives of this Master’s thesis research are:

1) To utilize wet okara to produce extruded cereal products.

2) To enrich extruded cereal products with okara.

3) To study the effect on isoflavone and dietary fiber contents of okara-
enriched extruded cereal products due to extrusion.
The following document includes a Literature Review (Chapter II), Materials
and Methods (Chapter III), Results and Discussion (Chapter IV), Conclusions

(Chapter V), Recommendations for Future Research (Chapter VI), and Appendices.

CHAPTER II
LITERATURE REVIEW

2.1 Soybean Seed Structure and Composition

There are three species of soybeans: Glycine ussun'ensis, Glycine max, and
Glycine gracilis. Of the three species, Glycine max is the most commonly grown.
There are many varieties that are grown and they typically vary in color, size, and
shape, but not in general structure. The soybean consists of three main
components: the hull, the cotyledon, and the hypocotyl, which constitute 8%, 90%,
and 2%, respectively, of the soybean (Waggle and Kolar 1979).

The cotyledon contains the major portion of proteins and lipids (Wolf 1996).
The fiber portions are concentrated in the hull and cotyledon (Lo 1989). The
isoflavones seem to be concentrated in the soybean hypocotyl with low to moderate
amounts in the cotyledon (Wang and Murphy 1994). Hughes and Murphy (1983)
reported that the protein contents among ten varieties of soybeans ranged between
39.4 - 44.1%. A large portion, 80 - 90%, of the protein content is made up of the
glycinin, B-conglycinin, and y-conglycinin storage proteins (Garcia et al 1997). Oil,
which makes up approximately 20% of the total soybean, is comprised of 88.1%
neutral lipids, 9.8% phospholipids, and 1.6% glycosides (Salunkhe et al 1982). The
main components of these lipids are oleic and Iinoleic fatty acids (Mahadvappa and

Raina 1978). Carbohydrates make up 33 - 35% of the soybean (Lucas and Riaz

1978) and consist mainly of polysaccharides and indigestible fiber (Weber et al

1993; Metelongo et al 1993).

2.2 Soymilk and Tofu Production

Soyfoods are typically divided into two categories: fermented and non-
fennented. Traditional non-fermented soyfoods include fresh green soybeans,
whole dry soyMans, soy nuts, soy sprouts, whole-fat soy flour, soymilk, soymilk
products, tofu, and okara (Golbitz 1995). Two of these products in particular have
found their way into mainstream Western culture; they are soymilk and tofu.

Soymilk is relatively simple to produce and is made by both small cottage
industries and large scale producers. According to Chen (1989), small scale
production requires a minimum of five steps, while large scale production requires
as many as eleven steps. Both large and small scale productions, however, follow
the same general steps. They first begin with cleaned whole soybeans which are
typically dehulled first. The beans are then soaked, typically in hot potable water, to
inactivate enzymes and soften the beans. If the beans are not soaked in hot water,
they are blanched after soaking in order to inactivate enzymes. The beans are then
ground with either hot or cold water using a stainless steel vertical or horizontal
grinder. The next step is to filter the slurry using a decanter centrifuge. The solids,
or “okara”, are removed and the liquid portion is cooked, deodorized to remove
volatile components, flavored, homogenized, pasteurized, and packaged (Ang et al
1985; Chen 1989; Golbitz 1995). Chen (1989) has also reported that many of the

previous steps may be combined in various ways to produce the best quality soymilk

possible. Ang et al (1985) reported the typical composition of soymilk to be 1.9%
protein, 0.66% fat, 0.10% ash, and 13.9% total solids.

Schaefer and Love (1992) define tofu as a gel-like precipitate obtained by
adding a calcium or magnesium salt to heated soymilk. There are many types of
tofu, but in North Ameriw the refrigerated type or aseptic type are most popular
(Murphy et al 1997). According to Watanabe and Kishi (1984), the process of
manufacturing regular tofu consists of twelve steps. Clean, whole soybeans are first
soaked in water for seven to fifteen hours. The soaked beans are drained and
ground. After grinding, 10 kg of water for every kg of soybeans is added to the
slurry and heated. A defoaming agent is added to the slurry and the sluny is
brought to a boil. Boiling continues for five minutes and then the mixture is filtered.
To the filtrate, which is soy milk, a coagulant (usually calcium sulfate) is added and
this mixture is stirred to disperse the coagulant. This mixture is allowed to settle,
while the residue from the filtrate, which is okara, is usually discarded. The
coagulated soymilk consists of curds and whey. The whey is drained off and
discarded, while the curds are wrapped in cloth and immediately formed into blocks.
These blocks are then soaked in cold water, the result is the final tofu product
(Smith et al 1960; Schaefer and Love 1992). The typical composition of tofu is 6.0%
protein, 3.5% fat, 1.9% carbohydrate, 0.6% ash, and 88.0% water (Smith et al

1960).

2.3 Okara and its Composition

Okara is the residue or soy pulp that remains alter the production of soymilk
and tofu. Although typically considered a waste product in the US, okara has been
shown to have many promising attributes. It has a typical composition that ranges
from 25.4% - 29.75% protein, 9.3% - 12.85% fat, 3.0% - 3.6% ash, 3.8% - 5.3%
carbohydrate, and 52.8% - 58.1% total dietary fiber (40.0% - 43.6% insoluble fiber
and 12.6% - 14.6 % soluble fiber), on a dry weight basis (Kronenberg et al 1984;
van der Riet et al 1989; Khare et al 1995). Wang and Murphy (1996) reported that
okara recovered after soymilk production contained a small amount of isoflavones
(25.9mgl717g okara, dry weight basis). Hackler et al (1963) reported that the
Protein Efficiency Ratio (PER) of okara was 2.71; in their study this was second only
to milk casein. This indicates that the protein in okara is of high quality. Despite the
obvious attributes, okara has seen only limited use as a food component for human

consumption.

2.4 Utilization of Okara

Every pound (4549) of dry soybeans made into tofu or soymilk generates
about 1.1 pounds (4999 or 2.5 cups) of firmly packed okara (approximately a 1:1
ratio) (Shurtleff and Ayogi 1979). Though nutritionally sound, okara has found only
little use as a human food source in the US. This is mainly due to the high
moisture content (greater than 80%) of okara and the cost associated with drying.
Furthermore, wet okara is highly susceptible to putrefaction (Noguchi and lsobe

1989). These problems have rendered the use of okara in the US. mainly to animal

feed and fertilizer. Okara’s use as human food in the US. has been limited to the
cottage industries whose small scale enables them to immediately incorporate okara
into baked goods or meat products. To date in the US, no large scale use for

okara has been found, thus a novel process to utilize okara is needed.

2.5 Dietary Fiber

In 1976, Trowell et al defined dietary fiber as the sum of Iignin and plant
polysaccharides that are not digested by endogenous secretions of the mammalian
digestive tract. This remains the most widely accepted definition of dietary fiber.
Currently, however, there is support to expand this definition to include
oligosaccharides that are resistant to hydrolysis by human alimentary enzymes (Lee
and Prosky 1995). In general, dietary fiber is composed of structural materials of
plant cell walls (including cellulose and non-cellulosic polysaccharides such as
hemicelluloses and pectic substances) and non-cellulosic polysaccharides (such as
gums and mucilages) (Southgate 1995). Soy fiber is a mixture of the cellulosic and
non-cellulosic structural components of the cell wall (Slavin 1991). The major
fractions are non-cellulosic and consist of acidic polysaccharides, arabinogalactan,
and arabinan chains; 11.0% are cellulosic components (Lo 1989). Acidic
polysaccharides are highly branched with a D-galacturonic and D-galactose
backbone interspersed with rhamnose, arabinose, xylose, and fructose; other non-

cellulosic polysaccharides are galactomannans and xylan (Aspinall et al 1967).

2.5.1 Health Benefits of Dietary Fiber
Dietary fiber has been shown to be associated with many positive health
benefits. Sacks (1993) stated that fiber-rich foods may confer protection against
cardiovascular disease. Commonly, dietary fiber is termed either soluble or
insoluble, based on its ability to solubilize in water. Soluble fiber is typically
associated with serum cholesterol lowering and changes in glycemic response
(Chen et al 1981; Ikeda 1993; Anderson et al 1994; Zhang et al 1994), while
insoluble fiber is primarily thought to affect the function of the large bowel by
decreasing transit time (Englyst et al 1995). Soy fiber, such as that from okara may
provide a readily available source of dietary fiber for use in a variety of food
products. Okara provides a source that has a good distribution of both soluble and
insoluble fibers, 12.6% - 14.6% and 40.0% - 43.6%, respectively (Kronenberg et al

1984), thus providing the health benefits of both fiber types.

2.5.2 Effects of Extrusion on Dietary Fiber

Extrusion is a popular processing technique that is used to make a variety of
products, most notably cereal and snack foods. Extrusion exposes the raw
materials to a number of mechanical forces including heat, compression, shear, and
swelling (Aoe et al 1989). The combination of these forces is likely to disorganize
the structure of these raw materials resulting in Changes in the materials.

In general, extrusion has a tendency to increase the soluble fiber fraction with
subsequent decreases in the insoluble fiber fractions, as the severity of the

extrusion conditions increases (Robertson and Eastwood 19881; Bjorck et al 1984;

Anderson and Clydesdale 1986; Caprez et al 1986; Siljestrom et al 1986; Ralet et al
1990; Wang et al 1993; Qian and Ding 1996). These observations may be a result
of disruption of covalent or non-covalent bonds in the carbohydrate and protein
moieties, leading to smaller soluble fragments (Wang et al 1993). It may also be
caused by the degradation of the amorphous regions of the fiber and the
compression of the crystalline regions (Qian and Ding 1996).

In terms of effects of extrusion on total dietary fiber, Bjorck et al (1984),
Ostergard et al (1989), and Lintas et al (1995) observed slight increases in total
dietary fiber. This effect is most likely due to the retrogradation of amylose forming
an enzyme-resistant starch upon cooling of the product (Englyst et al 1995). In
contrast, Wang et al (1993) observed slight decreases in total dietary fiber and
suggested the decreases were due to the disruption of covalent or non-covalent
bonds in the carbohydrate and protein moieties. On the other hand, Siljestrom et al
(1986) and Schweitzer and Reimann (1986) reported no changes in total dietary
fiber content. This broad spectrum of observations can most likely be attributed to
inherent differences in native composition of fiber from different sources, and to

differences in extnIsion conditions used.

2.5.3 Determination of Dietary Fiber

Dietary fiber is typically determined by one of two methods: ezymatic-
gravimetric or chemical. The enzymatic-gravimetric method, described by Lee et al
(1992), is used to determine total, insoluble, and soluble dietary fiber content in

food. The chemical method, described by Quigley and Englyst (1994), is used to

I0

determine the constituent sugars in the non-starch polysaccharides which make up
dietary fiber. The enzymatic—gravimetric method accounts for enzyme-resistant
starch content; on the other hand, the chemical method does not account for
enzyme-resistant starch. The inclusion of enzyme-resistant starch in the total
dietary fiber content is a source of debate, however according to Lee et al (1992)

the most widely accepted definition of dietary fiber includes enzyme-resistant starch.

2.6 Isoflavones

Phytoestrogens, compounds with weak estrogenic activity such as
isoflavones, coumestans, resorcyclic acid lactones, and lignans, are found in several
dozen plants eaten by humans, including soybeans and soybean products (Dweyer
et al 1994). Soybeans contain three types of isoflavones (daidzein, genistein, and
glycitein) and each exists in four chemical forms: as the aglycones, the glucosides,
the acetylglucosides (formed during processing by heat), and the malonylglucosides
(Figure 2.1) (Kudou et al 1991). Tsukamoto et al (1995) reported that the majority of
isoflavones found in soybeans are the glucosides genistin and daidzin, with the
malonylglucosides being the primary conjugates. Anderson and Wolf (1995) found
98.0% of the isoflavones present in soybeans to be in the glucoside form and 2.0%
to be in the aglycone form. Earlier studies showed the distribution of the isoflavones
to be 64.0% genistin, 23.0% daidzin, and 13.0% glycitin (Naim et al 1974). It has
been shown that different soybean varieties had similar isoflavone isomer
distributions but the total concentrations appeared to fluctuate due to genetics, crop

year, and growth location (Wang and Murphy 1994). In addition, different soy

11

 

 

 

Aglycones R1 R2 R3
Genistein OH H OH
Daidzein OH H H
Glucosldes R1 32 R3
Genistin O-glucosyl H OH
Acetyl Genistin O-glucosyl-6—OCOCH3 H OH
Malonyl Genistin O-glucosyl-6-OCOCHZCOOH H OH
Daidzin O-glucosyl H H
Acetyl Daidzin O-glucosyI-6-OCOCH3 H H
Malonyl Daidzin O-glucosyI-6-OCOCH2COOH H H

Figure 2.1 Structures of Isoflavone Isomers in Soybeans.

products contained different levels of the isoflavones and their distributions were
affected by particular processing techniques utilized to produce the product (Murphy

1982; Wang and Murphy 1994; Wang and Murphy 1996).

2.6.1 Health Benefits of Isoflavones

Recent health diet trends in the US. have included increased consumption of
soy foods. Much of this trend may be attributed to the increasing evidence that soy
foods contain isoflavones for which there is mounting evidence of a role in cancer
prevention. These cancer preventative properties have been demonstrated in
epidemiological studies (Goldin et al 1986; Aldercreutz et al 1992) and in animal
studies (Baggot et al 1990; Wei et al 1993; Hendrich et al 1994; Messina et al
1994). The pathways through which cancer prevention occurs are not known but
may be due to the ability of isoflavones to act as estrogen receptors (Peterson and
Bames 1991) and as free radical scavenging antioxidants (Wei et al 1995). The
amount of soy isoflavones that humans would need to consume in order to provide
an anticarcinogenic dose is approximately 1.5 - 2.0 mg (kg body weight)“day '1
(Heindrich et al 1994). Isoflavones have also been linked to reduction in LDL
cholesterol (Potter 1995) as well as the slowing down of atherosclerotic disease

(Wilcox and Blumenthal 1995).

2.6.2 Effects of Processing on Isoflavones
Most soyfoods undergo some type of processing, many of which involve heat

and/or fermentation. Wang and Murphy (1996) demonstrated through a mass

balance study that isoflavones were not destroyed by processing but the amount
and distribution of types of isoflavones in relation to the raw material was affected by
the type of processing. Products that undergo fermentation usually result in an
increase in the aglycones and a decrease in the glucosides (Lampe et al 1994;
Wang and Murphy 1996). This may occur through the hydrolysis of the malonyl
isoflavones into the glucosides and then into the aglycones by glucosidases in
soybeans (Matsuura and Obata 1993). Products that undergo heat treatment, most
commonly result in a partial conversion of the malonyl glucosides into their
respective acetyl glucosides (Wang and Murphy 1994). The malonyl isoflavones
have been shown to be heat labile and unstable at temperatures exceeding 100°C
(Pratt and Birac 1979). This conversion of the malonyl glucosides to acetyl
glucosides may be caused by the decarboxylation of the malonylglucosides.
Knowing the isoflavone content and distribution in soy products and byproducts, and
the changes they undergo during processing would be valuable information for

researchers pursuing the health benefits of isoflavones in processed foods.

2.6.3 Isoflavone Determination

Chromatographic methods have been used as early as 1974 for the
quantification of isoflavones. Niam et al (1974) utilized gas-liquid Chromatography to
quantify the content of daidzein and genistein in soybeans. West at al (1978)
developed a high-performance liquid chromatography (HPLC) method to quantify
genistein in soybeans. High-performance liquid chromatography methodology can

directly analyze free and conjugated forms of isoflavones without derivitization

14

(Wang et al 1990). unlike gas-liquid chromatography which requires derivitization
prior to analysis. Since 1978 various HPLC methods have been developed for the
determination of isoflavones in soybeans and soybean products (West et al 1978;
Eldridge 1982; Murphy 1982; Wang and Murphy 1994). Murphy (1981) investigated
various solvents for the extraction of isoflavones and coumestrol and found that
acetonitrile with water or hydrochloric acid was superior to all other extraction

solvents.

2.7 Soy Proteins

Hughes and Murphy (1983) reported that the protein contents among 10
varieties of soybeans ranged between 39.4% - 44.1%. It was shown that the
proteins were globular and heterogeneous, and were partitionable into four
centrifugal components with sedimentation coefficients of ZS, 78, 118, and 158
(Wolf and Briggs 1956). These components range in molecular weights from 8,000
to 480,000 Da (Wolf 1982). The 73 fraction is composed of mainly 7S globulin or
conglycinin and the 11S fraction is composed entirely of 118 globulin or glycinin.
The globulins, conglycinin and glycinin account for approximately 80% - 90% of the
proteins in soybeans (Klnsella 1984; Garcia et al 1997). The globulins are water
extractable proteins and can be precipitated out in acidic conditions (pH of 4.5 - 4.8)
(Fukushima 1991). Conglycinin is composed of three different subunits (a, B, and 7
subunits) that range in molecular weight from 44,000 to 59,000 Da, and glycinin

contains seven different acidic subunits (A1 - A7) and four basic subunits (B1 - B4)

15

which range in molecular weight fiom 37,000 to 45,000 Da and 19,000 to 22,000 Da,

respectively (Brooks and Morr 1985).

2.7.1 Amino Acid Compositions of Proteins in Soybeans and Okara

The nutritional value of proteins depends on amino acid composition.
Soybeans contain all nine essential amino acids required by humans (Garcia et al
1997). Moreover, it has been shown that the amino acid content in soybeans
exceeds the requirements for both children and adults (Steinke 1992; Lucas and
Raiz 1995). Hackler et al (1963) reported that the nutritional value of protein found
in okara was higher than in other soybean fractions. Wang and Cavins (1989)
showed that okara had an essential to total amino acid ratio that was comparable to
soymilk and tofu (Table 2.1). The amino acid composition of okara further proves its

value as a food source.

2.7.2 Processing Effects on Amino Acids

Hackler and Stillings (1967) reported that heat processing temperatures of
121°C resulted in decreases in cystine and tryptophan. Van Buren (1964) showed
that spray drying temperatures between 143°C and 316°C caused a decline in lysine
content. On the other hand, Khaleque and Wallace (1975) showed that processing
soymilk at temperatures between 98°C and 115°C resulted in no significant
changes in the amino acid composition. Jeunink and Cheftel (1979) reported that
some amino acid contents (methionine, asparagine, glutamine, and histidine) were

decreased and the cystine content increased after extrusion processing of soy

Table 2.1 Average Amino Acid Composition‘ (9 l 169 Nitrogen)

 

 

Amino Acid Soybeans Okara Soymilk Tofu
Aspartic Acid 12.61 11.63 11.91 11.70
Threonine 4.11 4.42 4.01 4.00
Serine 5.74 5.47 5.19 5.32
Glutamic Acid 19.76 17.71 19.61 19.26
Proline 5.53 5.66 5.33 5.47
Glycine 4.46 4.61 4.16 4.14
Alanine 4.49 4.36 4.14 4.11
Valine 3.73 5.28 4.88 4.99
Cystine 0.78 trace 0.03 trace
Methionine 1.34 1.67 1.59 1.43
lsoleucine 3.46 4.50 4.66 4.85
Leucine 7.90 8.31 7.94 8.32
Tyrosine 3.90 374 3.91 3.99
Phenylalanine 4.85 5.20 5.15 5.41
Lysine 6.19 6.36 6.08 6.41
Histidine 2.60 3.07 2.64 2.64
Arginine 8.64 8.61 8.65 8.52
Essential / Total 0.36 0.39 0.38 0.39

 

1(Wang and Cavins 1989)

17

protein, however none of these Changes were statistically significant. Changes in
the solubility of protein fractions and the amino acid composition of these fractions
after processing occur due to the involvement of particular amino acids in isopeptide
and/or disulfide crosslinking reactions (Stanley 1989). The amino acids of
importance to isopeptide crosslink formation are asparagine, aspartic acid,
glutamine, glutamic acid, histidine, lysine, and methionine; the sulfur-containing
amino acids, such as cysteine and methionine, are of importance to disulfide
crosslink formation (Stanley 1989). On the other hand, an increase in amino acid
contents of protein fractions after processing may be due to the unfolding of proteins
enabling more of the amino acid groups to become available to react with test
magent (Kwok and Niranjan 1995). What all of this may suggest is that spatial
relationships may be the controlling factor in the resulting effects on amino acids due

to processing (Stanley 1989).

2.8 Extrusion

Extrusion is a process that involves forcing a material to flow under a variety
of controlled conditions and then to pass through a shaped hole or slot at a
predetermined rate (Dziezak 1989). Extrusion is used to produce diverse cereal-
based products such as ready-to-eat cereals, snack foods, baked goods, and pet
foods (Clark 1986).

Extruders are typically put into two categories: single-screw extruders and
twin-screw extruders (Huber 1991). This discussion will be limited to twin-screw

extruders, which are commonly used for production of cereal-based products. Twin-

18

screw extruders, which contain two screws in the barrel of the extruder, are further
categorized by the type of screw configuration (conjugated or nonconjugated), the
relative direction of rotation (co-rotating or counter-rotating), and the degree of
intermeshing of the screws (intermeshing or non-intermeshing) (Harper 1989).

The following discussion will center on conjugated, co-rotating, and
intermeshing twin screw extruders. Conjugated refers to screws whose flights have
the same shape and dimension, and tightly fit the channels of the other screw with
minimum clearance (Martelli 1983). This is also referred to as self-wiping. Co-
rotating means the two screws rotate in the same direction. lntenneshing refers to
screws in which the distance between their centers is less than twice their outside
radius, therefore one screw engages with the other (Martelli 1983). The conjugated
and lntenneshing screw design allows the material in the barrel of the extruder to be
conveyed fonlvard with a positive pumping action, this being the basic difference
between single- and twin-screw extruders.

Several variables such as screw configuration, screw speed, material bed
rate, and temperature of the various heating zones of the barrel affect the extrusion
function, product quality, and product characteristics (Huber 1991). These variables
generate two effects: heating and shear. Upon heating and shearing cereal-based
materials, the macromolecules in food ingredients lose their native, organized
tertiary structure and form a continuous viscous dough (Harper 1986). The flow of
the material within the extruder screws aligns the large molecules in the direction of
the flow which exposes bonding sites and may lead to crosslinking which results in a

reformed structure (Harper 1986).

19

2.8.1 Screws and Paddles, Shear and Heat

Extruder screws are made up of a series of modular screw elements that can
be assembled in different configurations. The two basic types of elements used to
build screw configurations are feed screws, which can be either twin- or single- lead,
and kneading elements, which are also referred to as paddles and possess multiple
lobes. Twin-lead screws have two leads or flights which are 180° from each other.
The flight is a helical rib which wraps around the screw element. In contrast, single-
lead screw elements have only one lead. Twin-lead screw elements are used to
convey material while single-lead elements help to build pressure in the banal of the
extruder directly before the die exit.

Kneading elements are typically bi-lobed, and may be visualized as having
two blunt points on opposite ends. The kneading elements or paddles usually
occupy about a quarter of the length of the feed screws, and these sections are
considered areas where the majority of mechanical shear takes place. Mechanical
shear, in this thesis, refers to the physical disruption of material. The kneading
elements can be offset by 30°, 60°, or 90° with regard to the prior kneading element
and create significant shearing action (Harper 1986). The 30° offset is considered to
cause the least amount of mechanical shear and the greatest amount of material
conveyance; the 90° offset is considered to cause the greatest amount of
mechanical shear and the least amount of material conveyance. Kneading elements
can also be offset clockwise (forward) which causes forward conveyance of material
or counterclockwise (reverse) which causes backflow of material. Backflow of

material results in increased residence time of the material in the barrel and subjects

20

the material in the barrel of the extruder to the effects of shear and heat for a longer
period of time.

The number of kneading sections in a screw configuration, the offset degree
of the kneading elements and their forward or reverse orientation are primarily what
defines the overall degme of shear provided by the screw configuration. Shear is
partially responsible for physical and molecular disruption of the native state of the
raw material (Cumming et al 1972; Hager 1984; Harper 1986; Dahl and Wilma
1991). Shear also results .in the generation of mechanical energy which is
dissipated as heat (Harper 1986). Heat is also added externally to the barrel of the
extruder by either electricity or steam. Extrusion processing is unique from other
heat processes in that the material is also subject to shear (Wang et al 1993).

Conditions in the barrel of the extruder are considered more severe as heat
and shear increase, thus the more kneading elements, the greater the effects of
shear and heat on the material in the barrel of the extruder. The combination of
heat and shear have been shown to be responsible for decreasing viscosity (Mauser
and Wiedman 1989), increasing starch gelatinization (Bhattacharya and
Choundhurry 1994), affecting the solubility and bonding forces of proteins (Cumming
et al 1972; Hager 1984; Dahl and VIIlota 1991; Ferreira-Prudencia and Areas 1993),
and increasing the total and soluble fiber fractions (Bjorck et al 1984; Caprez et al
1986; Wang et al 1993). The therrnomechanical treatment that causes these
changes can be characterized by the determination of the specific mechanical
energy (Brent et al 1997), which is an indication of abusive conditions in the barrel of

the extruder.

21

2.8.2 Determination of Physical Characteristics of Extruded Products

Twin-screw extrusion is one of the most poplar processes used by the food
industry to produce expanded snack foods, ready-to-eat cereals, and pet food
(Chinnaswamy et al 1989). The microstructural properties of extruded products
depends on the influence of physical forces on chemical components (Stanley
1986). Various analyses have been used to determine the physical characteristiCs
that make up the microstructure of extruded products.

Radial expansion ratio has been used to determine the degree of expansion
of a product in comparison to the size of the exit die orifice (Moore et al 1990; Lue et
al 1991; Jin et al 1995). Bulk density values have been used to determine the
density of the products in relation to a specific volume (Hsieh et al 1991; Lue et al
1991; Jin et al 1995). High bulk density values are typically related to samples with
low radial expansion. Breaking strength has been used to determine how much
force is required to break extruded products. Jin et al (1995) related samples with
low expansion ratios to samples with high breaking strength values. Water
absorption and water solubility indices have been used to determine the ability of
extruded products to absorb water and to measure the amount of water soluble
material present in extruded products (Ralet et al 1990; Jin et al 1995), respectively.
The heat and shear from the extrusion process impact the water absorption and
water solubility indices. Rapid visco-analysis has also been used to determine the
pasting properties of extruded products (Ryu et al 1993; Whalen et al 1997).
Pasting properties are related to starch and the effects extrusion may have on

starch. It has also been shown that sucrose, shortening, non-fat dry milk, sodium

22

bicarbonate, fiber, protein, and fat affect the pasting properties of wheat flour (Ryu

et al 1993; Whalen et al 1997).

23

CHAPTER III

MATERIALS AND METHODS

3.1 Materials

Wet okara was obtained from American Soy Products Inc. (Saline, MI). The
okara was derived from the production of soymilk and was obtained immediately
after the soymilk production. The okara was placed in air-tight five gallon containers
(previously sterilized with a 10% chlorine bleach solution) and stored in a freezer (-
10°C) until use.

Soft wheat flour (Characteristics listed in APPENDIX l) was obtained from the
King Milling Co. (Lowell. Ml). Upon receiving, the four 50 pound sacks of flour were

combined in an air-tight bin and then stored in the freezer (-10°C) until needed.

3.2 Methods
3.2.1 Preparation of Formulations

Wet okara and soft wheat flour were combined in different proportions in
order to develop two formulations, designated A and B (Table 3.1). The wet okara
and flour were weighed in the appropriate proportions for the respective formulation
and combined in a large mixing bowl in a temperature (22°C) and humidity (45%)
controlled room. The materials were mixed at low speed for three minutes with a
HObart A-200 mixer (The Hobart MFG. 00., Troy, OH). The materials were mixed in

such a manner, to develop a formulation that had a particulate flow-like consistency.

24

Table 3.1 Proportions by Weight of Okara and Soft Wheat Flour Used for
Formulations A and B

 

 

 

 

Formulation Okara Soft Wheat Flour
_________ (°/o) _ (°/o)
A" 33.33 66.67
B2 40.00 60.00

 

‘Composition of A = 11.0% okara (d.b.), 33.0% water, and 56.0% flour (d.b.).
2Composition of B = 14.0% okara (d.b.), 37.5% water, and 48.5% flour (d.b.).

25

The particulate flow-like consistency was imperative in order to continuously deliver
a consistent amount of material to the barrel of the extruder. The materials were

mixed immediately prior to the extrusion process.

3.2.2 Extrusion Equipment

Extrusion was performed using an APV Baker MP19TC-25 (APV Baker,
Grand Rapids, MI) co-rotating and intermeshing twin-screw extruder with a 2kW
motor. The barrel diameter of this particular extruder was 19 mm and had a length
to diameter ratio (LzD) of 25:1. The barrel was a clam-shell style and had flighted
shafts that supported a modular screw system allowing the operator to design
unique screw configurations. There were five independently heated zones,
designated from feed port to die exit as zone 1 (Z1) through zone 5 (25). Electricity
provided the source of heat for the extrusion process. The particulate ingredients
were fed into the barrel of the extruder via a K-Tron K2M (K-Tron Corp., Pittman,
NJ) twin-screw volumetric feeder. Liquid ingredients could be fed into the barrel of
the extruder via a Brook Crompton E2 Metripump (Brook Crompton, Hudders Field,
England). The die used for this study consisted of a single circular orifice with a 3
mm diameter. A variable speed cutter, designed and built at Michigan State

University, was used to cut the extrudate to lengths of approximately 5 mm.

3.2.3 Extrusion Parameters

Two screw configurations were used for this study and were designated low

shear (LS) and high shear (HS) (Table 3.2). The LS configuration contained fewer

26

Table 3.2 Screw Elements1 Used for Low Shear and High Shear

Configurations

 

Low Shear Configuration (LS)

flgh Shear Configuration (HS)

 

8 D Twin Lead Feed Screws
7x30° Forward Kneading Elements
8 D Twin Lead Feed Screws
3x60° FonIvard Kneading Elements
3x30° Reverse Kneading Elements
2 D Single Lead Feed Screws
4x60° FonIvard Kneading Elements
3x30° Reverse Kneading Elements
2 D Single Lead Feed Screws

8 D Twin Lead Feed Screws
7x30° Forward Kneading Elements
4 D Twin Lead Feed Screws
4x60° Forward Kneading Elements
4x30° Reverse Kneading Elements
2 D Twin Lead Feed Screws
6x60° Forward Kneading Elements
4x30° Reverse Kneading Elements
1 D Single Lead Feed Screws
7x90° Kneading Elements
2 D Single Lead Feed Screws

1One kneading element = .25 D; One D = 19 mm

27

kneading element sections with more benign offsets than the HS configuration, thus
the HS configuration was considered more abusive. Two temperature profiles were
used for this study and were designated low temperature (LT) and high temperature
(HT) (Table 3.3). The comparatively higher temperatures of zones 3—5 for the HT
profile were considered more abusive than those delivered by the LT profile.

The two screw configurations and two temperature profiles were combined to
form four different extrusion conditions. The four conditions, in order of least
abusive to most abusive, were: low shear/low temperature (LS/LT), low shear/high
temperature (LS/HT), high shear/low temperature (HS/LT), and high shear/high

temperature (HSIHT).

3.2.4 Extrusion Process

The two formulations A and B were extruded in two replicates at each of the
four possible extrusion conditions. The existing water in the okara was the only
source of moisture for the formulations; no water was injected in the barrel of the
extruder for the extrusion process of either of the two formulations. The feed rate
for formulation A was 1.95 kg/hr and for formulation B was 3.71 kglhr. The
volumetric feeder was set at 1000 rpm for the delivery of the two formulations. The
screw speed for the barrel of the extruder was held constant at 100 rpm (500 rpm
was maximum for this extruder) for all extrusion work.

A flour/water sample was extruded in two replicates using the HSIHT

condition only. The flour/water sample (37.5% moisture) was extruded using the

28

Table 3.3 Temperature of the Five Heating Zones Used for the Low
Temperature (LT) and High Temperature (HT) Profiles

 

 

 

Temperature Zone 1 Zone 2 Zone 3 Zone 4 Zone 5
Profile (‘6) l°C) l°Cl (°C) (°C)
LT 40 60 125 135 145

HT 40 6O 1 3O 1 55 1 7O

 

29

traditional extrusion process of injecting water into the barrel of the extruder. The
flour feed rate was 3.0 kglhr (the volumetric feed rate was set at 925 rpm). The
screw speed was held constant at 100 rpm. Water was injected into the barrel of
the extruder at a rate of 0.5 kglhr; the liquid pump piston stroke was set at 11%.
Approximately two kg of extrudate was collected for each sample extruded.
The extrudates were placed on a table in a temperature (22°C) and humidity (45%)
controlled room until they could be placed in a forced-air oven. The extrudates were
allowed to dry overnight at 45°C in the forced-air oven. After drying, one kg of each
extrudate was ground using a Udy cyclone mill (Udy Corp., Fort Collins, CO) with a
1.00 mm mesh screen. Ground extrudate was placed in air-tight ziplock plastic bags
and stomd in the freezer (-10°C) until analyzed. The remaining one kg of each
extrudate was left unground, placed in air-tight ziplock plastic bags, and stored in

the freezer (-10°C) until analyzed.

3.2.5 Proximate Analysis of Samples

Proximate analysis of samples was performed on non-extruded okara, flour,
formulations A and B, extruded flour/water sample at the HSIHT condition . and
extruded formulations A and B for all conditions. Proximate analysis included
moisture, protein, ash, and fat analyses. Moisture, micro—Kjeldahl protein, and ash
analysis were determined utilizing AACC approved methods (1990).

Total fat analysis was performed utilizing the Tecator Soxtec HT 1043
extraction unit (Tecator, Hoganas, Sweden). The procedure used for fat analysis

was described by Foster et al (1992) with the following modifications: the samples

30

were not weighed into extraction thimbles and mixed with send, but were weighed
onto 11.0 cm diameter Whatman No.1 filterpaper (Whatman International LTD.,

Maidstone, England) and placed into the extraction thimbles.

3.2.6 Radial Expansion Ratio, Bulk Density, and Breaking Strength

Radial expansion ratios of the dried extrudates were determined by
measuring the cross sectional diameter of the unground extrudate divided by the die
orifice opening (3.0 mm). The cross sectional diameter of the extrudate was
measured with Vemeir calipers (Glogau and Co., Chicago, IL).

Bulk density has been defined as the weight of unground extrudate per unit
volume. Bulk density was determined by weighing pieces of extrudate after placing
them into a 100 ml graduated cylinder. The bottom of the cylinder was gently
tapped on a laboratory bench until there was no further reduction in sample volume.

Breaking strength has been defined as the maximum breaking force (N)
required to compress a sample of unground extrudate a given distance, divided by
the mass (9) of the sample. Breaking strength was measured utilizing a TA-XT 2
Texture Analyser (Stable Micro Systems, Surry, England) with a 0.5 inch diameter
stainless steel ball as the compression implement. Extrudate pieces from a sample
were placed into a cylinder with a 15 mm diameter opening and a 10 cm depth.
Sample weight was recorded and the sample then crushed a distance of 10 mm at a
rate of 1 mmlsecond. The peak force in Newtons (N) was recorded by Texture

Expert Software (Stable Micro Systems, Surry, England).

31

3.2.7 Water Absorption Index and Water Solubility Index

Water absorption index (WAI) has been defined as the weight of gel obtained
per gram of dry ground sample. Water absorption index was performed according
to AACC method 56-20 (1990).

Water solubility index (WSI) was defined by Jin et al (1995) as the
percentage of dry matter recovered after the supernatant from WAI analysis is
evaporated. The supernatant from the WAI analysis was decanted into a
preweighed plastic weighing boat and dried overnight in a forced-air oven at 45°C.
The calculation for WSI is as follows:

WSI (%) = [weight of dried supernatant I sample dry weight] x 100

3.2.8 Analysis of Pasting Properties

Pasting properties for non-extruded flour, non-extruded formulations A and B,
extruded flour/water sample at the HSIHT condition, and extruded formulations A
and B for all extrusion conditions were measured using the Rapid Visco-Analyser
(RVA) (Newport Scientific Pty. LTD., NSW, Australia). Five grams of each sample
(14% moisture basis) and 25 ml (plus moisture adjustment) of distilled water were
placed in an aluminum sample can as previously described by Deffenbaugh and
Walker (1989). A no.8 rubber stopper was inverted and placed over the opening of
the sample can in order to allow the sample can to be inverted ten times
immediately after the water was added to the sample. lnverting the sample helps to
prevent clumping and incomplete wetting of the material in the sample can. The

walls of the sample can were then wiped down with the stirring paddle blades and

32

 

placed into the RVA for analysis. The pasting profile used (Table 3.4) was
described by Ryu et al (1993). Four pasting parameters were measured: Peak
Viscosity, Breakdown, Setback, and Final Viscosity.

The term viscosity refers to the measurement of resistance on the RVA motor
and is expressed in rapid visco units (RVU). Peak viscosity is defined as the
maximum viscosity recorded by the RVA from the time the analysis begins until
immediately after the holding temperature is reached. Peak viscosities that occur
prior to the addition of heat may be considered a “cold paste”; peak viscosities
occurring after the holding temperature has been reached may be considered a “hot
paste” (Newport Scientific RVA Operation Manual 1995). Breakdown is defined as
the ability of a sample to withstand mechanical shear delivered by the RVA as the
holding temperature and stirring rate are held constant for a given period of time
(Newport Scientific RVA Operation Manual 1995). The setback value demonstrates
the degree to which retrogradation of starch occurs as the sample slurry is cooled
(Tipples 1980). The final viscosity is the viscosity of the sample recorded at the end

of the pasting profile.

3.2.9 Specific Mechanical Energy

Specific mechanical energy (SME) was determined on all extruded products.
The SME is defined as the mechanical energy required to obtain 1 kg of extrudate.
The SME was calculated using the following equation described by Brent et al
(1997):

SME = [screw rpm (run)lscrew rpm (rated))x [% torque/100] x [motor power/feed rate]

33

Table 3.4 Pasting Profile‘ Used for RVA Analysis

 

 

 

.,._T._i_me-lmin-)_ _ A. ,_ Iameerature,l°9) ._ $56919 Rate (rpm)
0 - 2 50 160
2 - 4 50 - 95 160
4 - 10 95 160
10 - 18 95 - 50 160
1Ryu et al (1993)

34

3.2.10 Dietary Fiber Analysis

The determination of dietary fiber followed the AOAC method 991.43 as
described by Lee et al (1992). The amounts of total dietary fiber (TDF) and
insoluble dietary fiber (IDF) were directly determined by analysis, while the soluble
dietary fiber (SDF) was indirectly determined by the difference (TDF - IDF = SDF).
For the determination of both TDF and IDF contents, duplicate samples were
weighed for each analysis. The fiber residue from one of the duplicate samples was
used to determine ash content and the fiber residue from the other duplicate sample
was used to determine protein content.

Two 1.000 g i .005 9 samples (sample wt.1 and sample M2) were used for
each analysis. The samples were suspended in a 2-(N-
Morpholino)ethanesulfonicacid-Tris(hydroxymethyl)aminomethane (MES-TRIS)
buffer (pH 8.2 at 24°C). This was followed by three consecutive enzymatic digestion
steps using a-amylase, protease, and glucosidase, sequentially. The digestion
steps were performed for both TDF and IDF samples. To determine IDF, samples
were filtered through a filtering crucible with a fritted disk (40pm - 60pm) immediately
after enzymatic digestion. To determine TDF, 230 ml of 95% ethanol was added to
samples after enzymatic digestion and allowed to sit for one hour. This is the
defining step for TDF because the addition of ethanol allows the SDF fraction to
precipitate out of the solution. After the precipitation period, the sample was filtered
in the same manner as the IDF sample. After filtering, all samples were dried
overnight in a forced-air oven at 130°C. The dried residues (residue wt.1 and

residue M2) were weighed and then one of the duplicate samples was ashed at

35

525°C and the other was analyzed for protein content using AACC method 46-13
(AACC 1993). Blanks consisting of reagents only were also analyzed. Both TDF
and IDF were determined with the following calculation:
{(A-B-C-D)IE}x 100

A = (residue wt.1 + residue wt. 2) l 2

B = protein wt.

C = ash wt.

D = blank wt.

E = (sample wt. 1 + sample wt. 2) I 2

3.2.11 Analysis of Isoflavones

Isoflavone analysis was based on the procedure developed by Wang and
Murphy (1994). One modification was that methanol was used as the mobile phase
instead of acetonitrile. The extraction of isoflavones for this study was based on a
1.000 9 dried sample of okara or equivalent such that when the formulated samples
were analyzed, the sample size was adjusted to equal 1.000 g of okara. The
weighed sample was first wet with 15 ml of 0.1 N HCI, after which 20 ml of absolute
methanol was added. The sample was then sonicated for 20 minutes and the
volume brought to 50 ml with absolute methanol. A 1.0 ml sample was drawn from
the 50 ml volume and centrifuged for five minutes at 10,000 x g. The supernatant
(extract) was then ready for analysis.

The isoflavone extracts were analyzed by High Performance Liquid

Chromatography (HPLC) with a Dionex AGP-1 Gradient Pump and Dionex EDM-2

36

Eluent Degas Module (Dionex Corp., Sunnyvale, CA), a Waters 486 Tunable
Absorbance Detector (Waters Corp., Milford, MA), and a Microsorb MV column (5p,
100A, C18 ) (Rainin Instrument Co., Wobum, MA). Samples were injected by a
Spectrasystem AS 3000 autoinjector (Spectrasystem, Freemont, CA) onto a 20pl
loop. Sample data were collected and analyzed with a Peaksimple Chromatography
Data System (Peaksimple, Las Vegas , NV). A linear gradient (Table 3.5) of two
solvents was used: solvent A was 89.9% water, 10% methanol, and 0.1% acetic
acid. solvent B was 99.9% methanol, and 0.1% acetic acid. Detection of isoflavones
occurs at 254 to 264 nm, and for this study the detector was set at 260 nm. The
total run time for each analysis was 48 minutes.

Six isoflavone peaks were identified, they were daidzin, genistin, malonyl
daidzin, malonyl genistin, acetyl genistin, and genistein (in order of elution). The
area under each peak was measured and pg isoflavone / g okara was calculated as
follows:

1.) area under peak I conversion factor = pg of isoflavone (20 pl injection)"
Conversion factors are (unit of area ( pg of isoflavone)"):
Daidzin = 3333.04
Genistin = 4380.7
Malonyl Daidzin = 2966.952
Malonyl Genistin = 4039.9
Acetyl Genistin = 4039.9
Genistein = 7100

2.) pg( 20pl)"x 50,000pI (wt. okara equivalent)“ = pg isoflavone (g okara)1

37

Table 3.5 Linear Gradient Program Used for HPLC Analysis of Isoflavones

 

 

 

 

Timelmin-I - ,-_,-,_, SolventA(%)“--.W-_-. SolventB(°/o) ..........
0.0 80 20
2.0 70 30
28.0 30 70
30.0 15 85
35.0 80 20
48.0 80 20

 

38

3.3 Statistical Analysis

All statistical analyses were performed with the Statistical Analysis System
(SAS Institute Inc., Cary, NC) under the General Linear Model procedure. One—way
and two-way analysis of variance (ANOVA) was performed and Fishers least

significant difference (LSD) was used for multiple mean comparison.

39

CHAPTER IV

RESULTS AND DISCUSSION

4.1 Proximate Analysis of Non-Extruded and Extruded Samples

Proximate analysis data for all of the non-extruded materials, extruded
flour/water sample at the high shear/high temperature condition, and extruded
formulations A and B for all conditions are listed in Table 4.1. The data for the
two formulations (A and B) reflect the combination of okara and flour in different
ratios. The moisture contents of the extruded samples listed were determined
after oven drying and grinding. The wide variation in moisture content may be a
result of differences in the length of time the extrudate samples were exposed to
ambient conditions during sample grinding. The moisture variation may also be
due to different amounts of enzyme-susceptible starch (gelatinized) in the
samples. Wang et al (1993) reported a higher percentage of enzyme-susceptible
starch increased water absorption. The extruded samples varied slightly in
protein and ash contents but showed no general trends compared to their
respective non-extruded sample. The protein, ash, and fat contents for all of the
extruded samples varied slightly among extrusion conditions but resulted in no
general trends. The fat content of all samples appeared to decrease slightly after
extrusion. Mercier (1980) suggested that monoglycerides and free fatty acids can

form complexes with amylose during extrusion cooking. The formation of these

40

Table 4.1 Proximate Analysis‘ Data for Non-Extruded and Extruded

 

Samples
Extrusion % Moisture % Protein % Ash % Fat
-___,_--..€=W<>.nditiensz. , '
Okara
Non-Extruded 77.65 24.21 4.06 9.83
Flour
Non-Extruded 10.57 8.54 0.56 1.03
F lourNVater(HS/HT) 3.55 8.53 0.57 0.97
Formulation A
Non-Extruded 33.00 9.75 0.96 1 .59
LSILT 7.78 9.63 0.98 1.56
LSIHT 6.42 9.76 0.98 1.54
HSILT 5.95 9.82 0.98 1.55
HSIHT 1.87 9.81 0.98 1.53
Formulation B
Non-Extruded 37.50 10.44 1 .07 1 .80
LSILT 6.48 10.42 1.10 1.74
LSIHT 7.13 10.39 1.09 1.74
HSILT 5.16 10.39 1.08 1.76
HSIHT 5.09 10.38 1.07 1.75

 

1All data expressed on a dry basis.
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

41

complexes may have made extraction of fat more difficult. Wang et al (1993)
also reported that fat content in whole wheat flour decreased after extrusion and

attributed the decrease to formation of complexes with amylose.

4.2 Physicochemical Analysis of Non-Extruded and Extruded Samples
4.2.1 Radial Expansion Ratio, Bulk Density, and Breaking Strength

Data for the radial expansion ratio, bulk density, and breaking strength for
extruded flour/water at the high shear/high temperature condition, and formulation
A and B extruded at all conditions are listed in Table 4.2. The expansion of
extruded products is generally attributed to starch content and type. Increasing
the content of certain ingredients has been demonstrated to affect the radial
expansion of extruded products. For example, increasing fiber content has been
demonstrated to decrease the radial expansion ratio (Moore et al 1990; Lue et al
1991; Jin et al 1995). Decrease in the radial expansion ratio as fiber content was
increased was also observed in the present study. The extruded flour/water
sample had the lowest fiber content and the greatest radial expansion ratio,
followed by formulation A and then by formulation B which had the greatest fiber
content and in general the lowest radial expansion ratio. Jin et al (1995)
suggested that increasing fiber content caused thickening of the cell walls and
decreased air cell size in the microstructure of the extrudate, resulting in
decreased radial expansion. There was also a decreasing trend in the radial
expansion ratio as extrusion conditions became increasingly more severe for

formulations A and B. This may be due to the breaking down of components into

42

Table 4.2 Radial Expansion Ratio, Bulk Density, and Breaking Strength

 

 

for Extruded Samples
Extrusion Radial Expansion Bulk Density Breaking
09nditi90§‘_ A . Ratio . _ . (911mm!) StrengtthlsL
Flour/Water
HSIHT 1 .57 48.85 33.33
Formulation A
LSILT 1.25 51.15 38.46
LSIHT 1.07 55.65 39.61
HSILT 1.00 58.30 42.54
HSIHT 0.98 59.40 45.15
Formulation B
LSILT 1 .00 55.60 40.09
LSIHT 0.96 57.40 44.28
HSILT 0.95 61.30 45.86
HSIHT 0.93 63.10 47.24

 

1Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

43

smaller particles, interfering with bubble expansion, reducing the extensibility of
cell walls and causing premature rupture of steam cells in the extrudate
microstructure resulting in decreased radial expansion (Guy 1985).

The bulk density values increased as radial expansion decreased (Table
4.2). This is due to the increased density of the extrudate microstructure as the
radial expansion decreases. Increased bulk density as radial expansion
decreased was also observed by other researchers (Hsieh et al 1991; Lue et al
1991; Jin et al 1995).

Jin et al (1995) demonstrated that breaking strength of extruded materials
is related to radial expansion. The results from the present study showed that
breaking strength increased as radial expansion ratios decreased (Table 4.2).
The increase in breaking strength is presumed to be due to the thickening of the
cell walls and decrease in air cell size. This may provide increased strength to

the extrudate microstructure and increased resistance to fracture.

4.2.2 Water Absorption Index and Water Solubility Index

Water absorption index (WAI) and water solubility index (WSI) for okara,
non-extruded flour, non-extruded formulations A and B, extruded flour/water at
the high shear/high temperature condition, and extruded formulations A and B for
all conditions are listed in Table 4.3. Water absorption and water solubility
indices were increased for all samples after extrusion. The WAI was most likely

increased after the extrusion process due to the pregelatinization of starch and

44

Table 4.3 Water Absorption Index (WAI) and Water Solubility Index (WAI)
for Non-Extruded and Extruded Products

 

 

 

Extrusion Conditions‘ 7‘ ___~___!_V_Afll_____m_ W wsr (%)
Okara
Non-Extruded 4.74 7.33
Flour
Non-Extruded 1 .91 4.84
Flour/Water (HS/HT) 7.64 8.25
Formulation A
Non-Extruded 2.09 5.83
LSILT 6.54 9.20
LSIHT 6.90 9.63
HSILT 6.70 9.56
HSIHT 7.55 9.91
Formulation B
Non-Extruded 2.13 6.12
LSILT 5.48 7.21
LSIHT 6.23 9.07
HSILT 6.72 8.47
HSIHT 6.72 8.50

 

1Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

45

an increased ability of fiber to absorb water compared to the respective non-
extruded sample. Pregelatinzed starch can absorb more water at ambient
temperature than raw intact starch and the extrusion process may break fiber into
smaller particles that have a greater surface area and thus are able to absorb
more water. The increased WSI contents were likely caused by increased
amounts of small soluble particles as a result of heat and shear from the
extrusion process, as described by Jin et al (1995) after extruding com meal at
various extrusion conditions.

The WAI for the extruded samples of both formulations A and B had
increasing trends as extrusion conditions became more severe. The increase in
WAI as extrusion conditions become more severe is contradictory to the findings
of other researchers who found WAI decreased with increased severity of
extrusion conditions (Jin et al 1995; Qian and Ding 1996). On the other hand,
Ralet et al (1990) and Wang et al (1993) found WAI of wheat bran to exhibit an
increasing trend as extrusion conditions became more severe, as observed in the
present study. Ralet et al (1990) proposed that interstices in fiber are emptied of
soluble material during extrusion and can quickly fill with water. Wang et al
(1993) proposed that a higher percentage of enzyme-susceptible (gelatinized)
starch in extruded samples contributed to higher water absorption. Variation in
extrusion conditions, types of raw materials, and combinations of raw materials
may account for the differences in WAI values observed in various studies. The
high fiber content of okara (53.25 % d.b.) may have been the major factor in the

WAI values observed in the present study, both prior to and after extrusion.

46

Water solubility indices increased as severity of extrusion conditions
increased for both formulations A and B. This trend was observed by other
researchers (Ralet et al 1990; Jin et al 1995). The increase in soluble
components, indicated by the WSI values in the present study, were a result of
the increase in temperature and mechanical shear as extrusion conditions
became more severe. The soluble particles may be derived from the soluble
starch component, smaller protein molecules, and other soluble components,

most likely soluble fiber of the okara.

4.2.3 Pasting Properties

Pasting properties obtained from the Rapid Visco-Analyser (RVA) for non-
extruded flour. formulations A and B, and the extruded flour/water sample at the
high shear/high temperature condition are listed in Table 4.4. Decreasing trends
were observed for peak viscosity, breakdown, setback, and final viscosity as fiber
content increased in non-extruded formulation A to formulation 8. VIscosity
increases are mainly attributed to swelled starch granules coming into contact
with each other (Rasper 1980). Continued heating causes starch granules to
rupture and leach out soluble amylose and amylopectin; the peak viscosity is the
equilibrium point at which swelling of starch and leaching out of soluble starch
components occur at equal rates (Rasper 1980). The decreasing trend in peak
viscosity coinciding with increasing fiber contents of the non-extruded samples
may be due to competition for water between starch and fiber components

making less water available for starch swelling and causing peak viscosity to

47

Table 4.4 Rapid Visco-Analyser oam‘ for Non-Extruded Formulations A
and B, Non-Extruded Flour, and Extruded Flour/Water at the

 

HSIHT: Condition
Sample Peak Breakdown Setback Final
_____ ___ ‘ Viscosity-.-W-- -- ___-. W -Yiscesitr- ._
Formulation A 431 282 214 361
Formulation B 401 273 102 319
Flour 489 372 236 403
Flour/Water(HS/HT) 482 451 46 77

 

1All data expressed in Rapid VISOO Units (RVU).
2HSIHT = high shear/high temperature extrusion condition.

48

decrease. Ryu et al (1993) observed decreased peak viscosity in wheat flour
based samples when various baking ingredients that competed with starch for
water were added. A proportionally decreased starch content as fiber content
was increased may also have contributed to the decreasing trend in peak
viscosity in the present study.

Breakdown represents the ease with which a sample can be broken down
by mechanical shear while constant temperature and stirring are maintained.
Small breakdown values represent more resistance to mechanical shear. The
decreasing trend in breakdown (Table 4.4) as fiber content increased in the non-
extruded samples may be due to the decreased starch contents and/or the
resistance to mechanical shear under the RVA conditions. The setback and final
viscosity values represent the reformation of the starch matrix as the sample is
cooled and is referred to as retrogradation. As retrogradation occurs, the
reformation of polymer-polymer bonds produce an increase in viscosity. There
was a decreasing trend for both setback and final viscosity values (Table 4.4) as
fiber content increased in the non-extruded samples of formulations A and B.

Figure 4.1 depicts viscograms of non-extruded flour and the extruded
flour/water sample at the high shear/high temperature condition. There is a
distinct shift of the peak to the left for the extruded sample as compared to the
non-extruded flour sample. This was observed for all extruded samples
compared to their respective non-extruded samples. The shift in the peak is
probably due to the pregelatinization of starch in the extruded samples which

enables starch granules to imbibe water and swell without the addition of heat.

49

 

' Non-Extruded Flour

 
 

Viscosity (RVU)

 

 

 

l I T I ’1 I l I I l [ l I U ‘ I I

, o 4 E g 12 . is ' an
Time (min)

 

Figure 4.1 Rapid Viscograms for Non-Extruded Flour Sample and
Extruded FlourNVater Sample at the High Shear/ High
Temperature Condition.

50

Breakdown value (Table 4.4) of the extruded flour/water sample reveals that the
starch granules in the sample were more susceptible to mechanical shear after
extrusion (higher breakdown values), suggesting that the starch granules were
more fragile. Setback and final viscosity reflect the retrogradation tendency of the
starch in the product (Tipples 1980). The decreased setback and final viscosity
values (Table 4.4) of the extruded flour/water sample may suggest that severe
damage has been done on the structure of starch decreasing the ability for
retrogradation.

Table 4.5 lists values for the pasting parameters of extruded formulations
A and B. The peak viscosity and breakdown values for the formulation A sample
extruded at the high shear/high temperature condition was significantly higher
(p30.05) than those of the other extruded formulation A samples. On the other
hand, there were no significant differences (p30.05) among peak viscosity and
breakdown values of formulation B, except for the sample extruded at the low
shear/low temperature condition, which was significantly higher. The peak
viscosities for formulation B had a decreasing trend as extrusion conditions
became more severe. The setback and final viscosity values for the extruded
formulation A samples varied in their significant differences, however, no
significant differences were observed among the extruded formulation B samples.
regardless of the extrusion conditions.

The results for the RVA study of the extruded formulations are difficult, at
best, to attribute to any one component of the two formulations in this study.

Rapid visco-analyser results of extruded products may be affected by starch,

51

Table 4.5 Rapid Visco-Analyser Data1 for Extruded Formulations A and B

 

Samples
Extrusion Peak Viscosity Breakdown Setback Final
___-929919.903? t - - V Eli-13999.31--.

Formulation A
LSILT 2703 232[3 53" 90"
LSIHT 3153 284° 458 77C
HSILT 289B 2543 54" 89“
HSIHT 449" 417" 50"8 82BC
LSD3 111.31 112.92 5.88 8.05

Formulation B
LSILT 383" 324" 65" 109"
LSIHT 285B 246B 53" 92"
HSILT 304B 266"‘3 58" 97"
HSIHT 2543 219'3 51" 86"
L303 54.89 68.29 32.48 25.25

 

1AII data expressed in Rapid VISC Units (RVU)
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature
3Least Significant Difference: samples sharing the same letter within a column for
Formulation A or Formulation B are not significantly different at 0t=.05

52

starch composition, starch-lipid interactions, starch-protein matrix, and soluble
fiber components (Whalen et al 1997). These various effects become
increasingly complicated when taking into account not only the possible
interactions of starch, protein, lipids, and fiber, but also the difference in the
sources of these components (soft wheat flour and okara). Gomez and Aquilera
(1984) proposed a model of starch degradation during extrusion in which starch
exists in three main states (raw. gelatinized, and dextrinized) and three
intermediate states (mechanically damaged, free polymers, and oligosacharides).
They observed decreased peak viscosity on amylograms as the content of
dextrinized starch increased in extruded corn starch samples. Ryu et al (1993)
demonstrated that adding shortening powder in extruded wheat flour products
decreased both peak viscosity and breakdown values on rapid viscograms. They
suggested that lipid-amylose complexes caused inhibition of starch hydration
resulting in decreased swelling of starch granules decreasing the viscosity and
susceptibility to mechanical shear. Whalen et al (1997) suggested that starch-
protein matrix formation in extruded corn flour resulted in increased resistance to
breakdown and high setback values in rapid viscograms. It is presumed that
fiber, and soluble fiber in particular, can result in decreased peak viscosity due to
competition with starch for water. Determining what particular interactions may
have occurred for both formulations A and B and their effects on the rapid

viscograms were beyond the scope of the present study.

53

4.2.4 Specific Mechanical Energy

The specific mechanical energy (SME) for the extruded formulations A and
B and the extruded flour/water sample at the high shear/high temperature
condition are listed in Table 4.6. The SME was higher overall for formulation A
than for formulation B. This was expected due to the lower moisture content and
greater proportion of flour in formulation A than formulation B resulting in a higher
apparent viscosity for formulation A The higher viscosity material required more
energy to turn the screws in the extruder and this was reflected in higher SME
values. Martelli (1993) determined that SME increased with increasing dough
mass viscosity. The SME decreased as temperature increased from the low to
the high temperature profile for both screw configurations and for both
formulations A and B. The decreased SME can be attributed to a decrease in the
viscosity of the material in the barrel of the extruder as the temperature was
increased. Weidmann (1990) observed a similar effect with SME values
decreasing when barrel temperature was increased during the extrusion of wheat
flour

Changing screw configuration from low to high shear was expected to
increase SME due to the slowing down of the forward flow of material in the
barrel of the extruder and the increasing energy required to turn the screws of the
extruder, however, the opposite effect was observed in the present study. The
unexpected decrease may have been a result of the fiber in the okara. The high
shear configuration may have broken the fiber into smaller fragments, increasing

the surface area of the fiber and its lubricating effect, and decreasing the torque

54

Table 4.6 Torque, Feed Rate, and Specific Mechanical Energy (SME) Data
for Extruded Formulations A and B and Extruded Flour/Water at

 

the HSIHT Condition
Sample Extrusion Torque Feed Rate SME
- __ ._._.§_9r!djti,9_n§1 _. W651-.-” Jest)...- --.-(!t!Y_h/-89L
Formulation A LSILT 42.5 1.95 0.0876
Formulation A LSIHT 37.5 1.95 0.0773
Formulation A HSILT 40 1.95 0.0824
Formulation A HSIHT 32.5 1.95 0.0670
Formulation B LSILT 35 3.71 0.0378
Formulation B LSIHT 32.5 3.71 0.0351
Formulation B HSILT 35 3.71 0.0378
Formulation B HSIHT 30 3.71 0.0324
FlourNVater HSIHT 30 3.00 0.0399

 

1Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

55

values requimd. The SME calculation is based on percent torque values and the
motor speed (Brent et al 1997); if torque decreases while the screw speed and
material feed rate are maintained constant, the SME will decrease. The torque
value for extruded flour/water at the high shear/high temperature condition did not
differ from that of formulation 8 and was only 2.5% lower than formulation A
extruded at the same conditions, however, the SME for the flour/water sample
was not similar to SME values for either formulation A or B. This is due to the
feed rate of the flour/water sample which was 3.0 kglhr compared to 1.95 kglhr

for formulation A and 3.71 kglhr for formulation B.

4.3 Dietary Fiber Contents in Non-Extruded and Extruded Samples

The total dietary fiber (TDF), insoluble dietary fiber (IDF), and soluble
dietary fiber (SDF) contents of the non—extruded samples are listed in Table 4.7.
The TDF contents of non-extruded formulations A and B increased 247 percent
and 262 percent, respectively, compared to TDF contents of non-extnlded flour,
as a result of the combination of okara and flour. This demonstrates that okara
can be used to enrich the TDF content of soft wheat flour. The IDF contents of
the two formulations increased, as expected, due to the combination of okara and
flour. Formulation A resulted in a slight increase in SDF content due to the
combination of okara with flour, however formulation B which was also expected
to increase in SDF content actually decreased slightly. The decrease in SDF

content for formulation B was within the acceptable range of variation for

56

Table 4.7 Insoluble, Soluble, and Total Dietary Fiber Contents1
of Non-Extruded Okara, Formulations A and B, and Comparison
Between Non-Extruded Flour and Extruded FlourNVater at the

 

 

 

 

 

HSII-IT2 Condition
Sample _____ % Insoluble ____- % Soluble . ___-___ % Total

Okara 43.27 9.98 53.25
Formulation A 7.66 2.08 9.74
Formulation B 8.84 1.46 10.30
Flour 2.22" 1.71" 3.93"
FlourNVater (HS/HT) 1.383 2.13B 3.503
LSD3 0.49 0.35 0.32

 

1All data expressed on a dry basis
2Extrusion condition used (screw configuration/temperature profile):
HSIHT = high shear/high temperature
3Least Significant Difference: samples sharing the same letter within a column are

not significantly different at 0t=.05

57

the dietary fiber assay (personal communication, Bennink, M.R., Michigan State
University).

The TDF, IDF, and SDF contents were significantly different (p50.05) for
the flour/water sample after extrusion at the high shear/high temperature
condition (Table 4.7). Wang et al (1993) also observed decreased TDF and IDF
contents and increased SDF contents after extrusion of whole wheat.

Table 4.8 lists the TDF, IDF, and SDF contents for non-extruded and
extruded formulations A and B. The TDF content for the formulation A sample
extruded at the low shear/low temperature condition was significantly lower
(ps0.05) than that of the non-extruded formulation A sample. This was the only
extruded sample to significantly differ from the non—extruded formulation A
sample. Based on the premise that the low shear/low temperature condition is
the most benign of the extrusion conditions used in the present study and the
least likely candidate to cause a decrease in TDF content, the decreased TDF
content may be attributed to variation in sampling. The TDF content for the
formulation B sample extruded at the high shear/low temperature condition was
the only one significantly different (higher; p30.05) than that of the non-extruded
formulation B sample. Englyst et al (1995) suggested the increase in TDF
contents of starchy foods, such as those containing white flour, after heat
processing is due to the formation of enzyme-resistant starch which takes place
during the cooling of the product and is a result of retrogradation.

The IDF contents of the extruded products of both formulations A and B

decreased for all extrusion conditions compared to their respective non-extruded

58

Table 4.8 Insoluble, Soluble, and Total Dietary Fiber Contents1 for
Non-Extruded and Extruded2 Formulations A and a

 

 

 

 

 

Sample __ _% Insoluble __ % Soluble W % Total
Formulation A
Non-Extruded 7.66" 2.08'3 9.74"
LSILT 6.428 2.823" 9.248
LSIHT 6.638 3 16" 9 79"
HSILT 5.81C 3:69" 9.50"'3
HSIHWT 5.71c 3.72" 9.43"B
LSD 0.56 0.98 0.48
Formulation B
Non-Extruded 8.84" 1.46D 10.30B
LSILT 8.50: 2'25; 10.72:
LSIHT 7.31 3.1 10.50
HSILT 8.31" 3.09'3 11.40"
HSIHT 672° 3.64" 10.368
LSD3 0.56 0.48 0.43

 

1All data expressed on a dry basis
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature
3Least Significant Difference (LSD): samples sharing the same letter within a
column for Formulation A or Formulation B are not significantly different at 0t=.05

59

formulation. The IDF contents were decreased significantly (p30.05) at all
extrusion conditions for formulation A, however, only the formulation B samples
extruded at the low shear/high temperature and high shear/high temperature
conditions resulted in significant decreases (ps005). Changing the temperature
profile from low to high temperature had a significant effect (p=0.0007) on the IDF
contents for the extruded formulation B samples; on the other hand, Changing
screw configuration from low to high shear had a significant effect (p=0.0086) on
the IDF content of the extruded formulation A samples. It appears that the
extrusion conditions required to affect the IDF contents are formulation
dependent

The SDF contents for extruded formulation A and B samples increased
significantly (ps0.05) for all extrusion conditions compared to their respective non-
extruded formulations with the exception of the formulation A sample extruded at
the low shear/low temperature condition. The nonsignificant increase for the low
shear/low temperature extruded formulation A sample may have been significant
if the TDF content for this sample were not significantly decreased. No one
extrusion parameter had a significant effect on the SDF content for extruded
formulation A samples, however, both temperature and screw configuration had
independent but significant effects (p=0.0067 and p=0.0104, respectively) on the
SDF contents of formulation B.

In the present study, the IDF contents decreased and the SDF contents
increased after extrusion for flour/water and formulations A and B as compared to

their respective non-extruded samples. The IDF contents for formulations A and

60

B tended to decrease as extrusion conditions became more severe; on the other
hand, the SDF contents tended to increase. Other researchers reported similar
findings (Robertson and Eastwood 1981; Bjorck et al 1984; Anderson and
Clydesdale 1986; Caprez et al 1986; Siljestrom et al 1986; Ralet et al 1990;
Wang et al 1993; Glen and Ding 1996). Wang et al (1993) suggested that the
disruption of covalent and non-covalent bonds in the carbohydrate and protein
moieties, leading to smaller soluble components results in increased SDF and
decreased IDF contents during extrusion. Recently Qian and Ding (1996)
suggested that decreased IDF and increased SDF contents after extrusion are a
result of the degradation of the amorphous regions and the compression of the
crystalline regions in fiber. In general there were no significant changes in total
dietary fiber contents after extrusion with the exception of the low shear/low
temperature formulation A sample and the high shear/low temperature
formulation B samples. Various effects on TDF contents due to extrusion have
been observed. Bjorck et al (1984), Ostergard (1989), and Lintas et al (1995)
observed increased TDF contents and attributed them to formation of enzyme-
resistant starch. Siljestrom et al (1986) and Schweitzer and Reimann (1986)
reported no Changes in TDF contents after extrusion. Wang et al (1993)
observed decreased TDF contents after extrusion and attributed this to
components being broken down into smaller soluble components. The variation
in results observed for TDF contents after extrusion are most likely due to the
inherent differences in the native compositions of fibers from different sources

and to variations in the extmsion conditions used.

61

4.4 Isoflavone Contents in Non-Extruded and Extruded Samples

There were no detectable levels of isoflavones in the non-extruded flour
and the extruded flour/water sample at the high shear/high temperature extrusion
condition (data not shown), based on spectroscopy at 260 nm. Six isoflavone
compounds were identified in okara and the non-extruded and extruded samples
of formulations A and B, confirming that okara can be used to enrich the
isoflavone contents of soft wheat flour. The six isoflavone compounds were, in
order of elution: daidzin, genisitin, malonyl daidzin, malonyl genistin, acetyl
genistin, and genistein (Figure 4.2). The levels of detected isoflavones and total
detectable isoflavones for non-extruded samples of formulations A and B are
listed in Table 4.9.

The glucoside, malonyl, and acetyl forms of daidzin and genistin combined
represent 90-98 percent of the total detectable isoflavones in okara and the non-
extruded formulations A and B. Wang and Murphy (1994) and Anderson and
Wolf (1995) also found greater than 90 percent of the total detectable isoflavones
to be a combination of the glucoside, malonyl, and acetyl isoflavones in soy and
soy products. Malonyl daidzin and malonyl genistin were the most abundant of
the isoflavone compounds in okara and the non-extruded samples of formulations
A and B. There was little difference in the levels of total detectable isoflavones
among the samples. The two formulations exhibited Changes in the distribution of
the isoflavones in comparison to the okara sample. Non-extruded samples of
formulations A and 8 had decreased levels of daidzin, genistin, malonyl daidzin,

and malonyl genistin and increased acetyl genistin and genistein levels as

62

.92:

 

Flgure 4.2 HPLC Chromatogram for Okara Deplctlng Detected Isoflavones
In Order of Elutlon.

63

Table 4.9 Individual and Total Detected Isoflavone Contents1 of
Non-Extruded Samples

 

 

Compound Detected .. Okara .FOmeathA FormulationB
Daidzin 108 83 90
Genistin 141 65 45
Malonyl Daidzin 399 360 360
Malonyl Genistin 489 465 447
Acetyl Genistin 80 142 147

Genistein 24 103 1 16
Total Detected 1241 1219 1205

 

1All results expressed on a pglg dried okara basis

64

compared to the okara sample. A possible explanation for the redistribution of
isoflavones in the two formulations may be an interaction between okara and flour
and/or fermentation of the formulations during mixing that may have caused the
conversion of the glucoside and malonyl isoflavones to the acetyl and aglycone
isoflavones via a hydrolysis reaction with glucosidases already present in the
formulations. Wang and Murphy (1994) observed the occurrence of similar
distributional changes in fermented soy products.

Tables 4.10 and 4.11 list levels of the six detectable isoflavones and the
total detectable isoflavones for the non-extruded and extruded formulations A and
B samples, respectively. Total detectable isoflavone levels for both formulations
A and 8 decreased alter extrusion for all extrusion conditions compared to their
respective non-extruded formulation. The total detectable levels of isoflavones
in formulation A samples extruded at the low shear/high temperature and the high
shear/low temperature conditions decreased significantly (p50.05). The total
detectable levels of isoflavones in formulation B samples extruded at the low
shear/high temperature and high shear/high temperature conditions were
decreased significantly (p50.05). The decreased total detectable isoflavone
levels after extrusion may be due to degradation of the isoflavone compounds or
conversion to non-detected isoflavone compounds as a result of the heat and
shear generated from the extrusion process.

The glucoside, malonyl, and acetyl forms of the isoflavones represent 96-
98 percent of the total detected isoflavones after extrusion of the formulation A

and B samples. This is in agreement with other findings (Wang and Murphy

65

Table 4.10 Individual and Total Detected Isoflavone Contents1
for Non-Extruded and Extruded2 Formulation A Samples

 

Compound Non-Extruded LSILT LSIHT HSILT HSIHT Lso“
Wgetested

 

 

 

Daidzin 83“ 171““ 198“ 155“ 261" 34.112
Genistin 66“ 177“ 202“ 163“ 298" 86.373
Malonyl Daidzin 360" 302“ 145“ 263“ 131“ 47.357
Malonyl Genistin 466" 327“ 150D 267“ 146D 56.774
Acetyl Genistin 142“ 121“ 238" 119“ 212" 36.850
Genistein 104" 36“ 36“ 32“ 29“ 17.585
Total Detected 1219" 1133"“ 963“ 1002“ 1072"“ 151.490

 

1All results expressed on a pglg dried okara basis
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature
3Least Significant Difference (LSD): samples sharing the same letter in a rmv
are not significantly different at 01:05

66

Table 4.11 Individual and Total Detected Isoflavone Contents‘
for Non-Extruded and Extruded2 Formulation B Samples

 

Compound Non-Extruded LSILT LSIHT HSILT HSIHT Lso“
_ __ -_ Detected ..

Daidzin 90“ 153" 201" 192" 198" 47.829
Genistin 45“ 188" 180" 214" 226" 116.86
Malonyl Daidzin 361" 335" 155“ 273“ 138“ 63.499
Malonyl Genistin 448" 358“ 181“ 313“ 155“ 57.709
Acetyl Genistin 147" 104“ 181" 104“ 180" 41.814
Genistein 1 16" 36“ 35“ 32“ 40“ 27.398
Total Detected 1205" 1 175" 935“ 1133" 967“ 84.039

 

1All results expressed on a pglg dried okara basis
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature
°Least Significant Difference (LSD): samples sharing the same letter in a row
are not significantly different at (1:05

67

1994; Anderson and Wolf 1995). Extrusion of formulations A and B resulted in
distributional changes in the detected isoflavone compounds compared to their
respective non-extruded formulation. Distributional Changes among isoflavone
compounds after processing have been reported (Pratt and BiraC 1970; Matsuura
and Obata 1993: Lampe et al 1994; Wang and Murphy 1994; Wang and Murphy
1996). The glucosides, daidzin and genistin, significantly increased (p30.05) after
extrusion for both formulations A and B regardless of the extrusion conditions.
Wang and Murphy (1984) also observed increased glucoside isoflavone contents
in soy protein isolate compared to intact soybeans from the same source. A
possible cause for this Change could be a loss of the malonyl group resulting in
the respective glucoside form of the isoflavone. The malonyl group may have
been cleaved off as a result of heat and/or shear generated by the extrusion
process.

Malonyl daidzin and malonyl genistin contents decreased after extrusion
under all extrusion conditions for both formulations A and B. Decreases in
malonyl daidzin and malonyl genistin contents were significant (p50.05) at all
extrusion conditions except for the malonyl daidzin content of formulation B
extruded at the low shear/low temperature condition. The largest decreases in
both malonyl daidzin and malonyl genistin occurred at the high temperature
extrusion conditions for both formulations. The decreased malonyl genistin
contents at the high temperature conditions were offset by significantly increased
(p50.05) acetyl genistin contents for formulation A and non-significant increases

for formulation B after extrusion. Farmakalidis and Murphy (1985) reported that

68

decreased malonyl isoflavone contents were offset by increased acetyl isoflavone
contents after toasting defatted soy flakes. In addition, Wang and Murphy (1984)
proposed that malonyl isoflavones may be converted into their respective acetyl
isoflavones via the decarboxylation of the malonyl isoflavone. Malonyl
isoflavones are heat labile and unstable at temperatures exceeding 100°C (Pratt
and Birac 1979). Temperatures used in the present study exceeded 100°C
during extnlsion using both the low and high temperature profiles and may
explain the decreased malonyl daidzin and malonyl genistin contents in the
extruded formulation A and B samples.

The aglycone, genistein, was significantly decreased (p30.05) after
extrusion regardless of extrusion conditions for both formulations A and B. No
literature was found to support decreased genistein contents after extrusion
processing, however, it is presumed that genistein is either degraded or

converted to forms of isoflavones nondetectable at 260 nm.

69

CHAPTER V

SUMMARY AND CONCLUSIONS

Two formulations of wet okara and soft wheat flour (A and B) were
extruded at four screw configuration/temperature profile conditions. Extrusion
conditions affected the physical characteristics of the extrudates. Radial
expansion ratio decreased, and bulk density and breaking strength values
increased as extrusion conditions became more severe for both formulations A
and B. Increasing trends were observed for both water absorption index and
water solubility index for both formulations as severity of extrusion conditions
increased. Pasting parameters for extruded products showed the complexity of
the formulations. Peak viscosity and breakdown values resulted in an increasing
trend for formulation A as the extrusion conditions became more severe; on the
other hand, formulation B (which had proportionally more okara and less flour by
weight) resulted in an opposite trend. Low setback and final viscosity values
were observed for all extruded samples demonstrating that the starch granules in
the samples had sustained irreparable damage. Specific mechanical energy
values tended to decrease as extrusion conditions became more severe for both
formulations A and B.

Combining okara with $011 wheat flour resulted in increased protein, dietary
fiber, and isoflavone contents compared to soft wheat flour alone. The extrusion

process had little effect on the protein contents of the extrudates of formulations

70

A and B. Variable effects were observed for the total dietary fiber contents of the
two formulations’ extrudates. The extrusion process consistently decreased the
insoluble dietary fiber content and increased the soluble dietary fiber content of
both formulations as the extrusion conditions became more severe. Total
detectable isoflavone contents were decreased at all extrusion conditions
compared to their respective non-extruded formulation. Changing the extrusion
temperature profile from low to high resulted in decreased malonyl daidzin,
malonyl genistin, and genistein contents, while acetyl genistin content increased
for both formulations A and B.

The overall results demonstrate that by utilizing 'a novel twin screw
extrusion process to cook, sterilize, and remove the major portion of water, wet
okara could successfully be used to make extruded products. Changing the
extrusion conditions resulted in varied physical Characteristics for the extruded
products of the two formulations. Combining wet okara with soft wheat flour
enriched the protein, dietary fiber, and isoflavone contents. The extrusion
process had little effect on the protein content of the extrudates, however, dietary
fiber and isoflavone contents were affected. Extrusion decreased insoluble
dietary fiber and increased soluble dietary fiber contents. The extrusion process
decreased total detectable isoflavone contents and altered the distribution of the

six detectable isoflavones.

7I

CHAPTER VI

SUGGESTIONS FOR FUTURE RESEARCH

Wet okara was successfully used to produce extruded products and enrich
the protein, dietary fiber, and isoflavone contents of soft wheat flour, making it a
desirable component for use in commercially extruded products. Commercial
formulations, however, contain other ingredients such as sugar and shortening
that not only complicate the extrusion process, but can have adverse effects on
extrudate color, texture, and flavor. In order for the use of wet okara to be
considered by product manufacturers, it is necessary to develop a study that
demonstrates wet okara can be used in a scaled-Up manufacturing process to
successfully produce commercially acceptable extruded products.

The first phase of the study would be to develop formulations utilizing not
only wet okara, but also sugar and shortening at levels used in commercial
formulations, scale up the extrusion process from laboratory to pilot scale, and
extrude these formulations under various conditions. A second phase of the
study would include analyzing the physicochemical properties of the extruded
products and comparing those properties to the phyicochemical properties of
commercial products. This phase would also include microscopy analysis of the
extrudates in order to study the microstructure. The microstructure is largely
responsible for the textural Characteristics of extruded products. The last phase

of this study would be a sensory evaluation of the extruded products made with

72

the wet okara in order to determine the consumer acceptability of the texture,
flavor, and color of these products.

Utilizing wet okara in a commercial extrusion process would add value to
this currently valueless by-product in North America. Furthermore, utilizing okara
in commercial extnlded products would provide consumers with a source of

dietary fiber and isoflavones.

73

APPENDICES

74

APPENDIX I

Flour Characteristics Determined by 1Farinograph Constant Flour Weight
Method

 

Flour Consistency Water Arrival Stability Mixing

 

Weight (BU) Absorption Time (minutes) Tolerance
(g) (%) (minutes) Index
(BU)
48.08 510 50.80 1.00 1.00 90

 

1Flour weight for the farinograph constant flour weight method was
adjusted for a 14% moisture basis. Consistency and Mixing Tolerance
Index are expressed in Brabender Units (BU).

75

APPENDIX II

Raw Data‘ for Moisture Analysis of the Non-Extruded and Extruded
Samples

 

 

 

Extrusion ConditionsT Moisture 1 Moisture 2 Moisture 3
(‘96) ('56) (96)
Okara
Non-Extruded 77.77 77.66 77.57
Flour
Non-Extruded 10.84 10.72 10.15
FlourNVater Rep 1(HSIHT) 3.93 4.17 4.14
FlourNVater Rep 2(HSIHT) 3.02 3.04 2.99
Formulation A
Non-Extruded 32.50 34.19 32.23
LSILT Rep 1 8.24 8.50 8.48
LSILT Rep 2 6.98 7.24 7.21
LSIHT Rep 1 5.47 5.95 5.87
LSIHT Rep 2 7.08 7.03 7.13
HSILT Rep 1 7.10 7.61 7.68
HSILT Rep 2 4.27 4.37 4.64
HSIHT Rep 1 1.97 1.98 1.96
HSIHT Rep 2 1.78 1.77 1.75
Formulation B

Non-Extruded 37.81 38.91 35.78
LSILT Rep 1 6.70 6.97 7.12
LSILT Rep 2 5.85 6.09 6.11
LSIHT Rep 1 6.27 6.67 6.76
LSIHT Rep 2 8.02 7.46 7.59
HSILT Rep 1 4.24 4.21 4.26
HSILT Rep 2 5.89 6.10 6.24
HSIHT Rep 1 4.52 5.16 5.03
HSIHT Rep 2 4.86 5.15 5.19

 

1All data for extruded samples were determined after the samples were dried and
ground.
2Extrusion conditions used (screw configuration/temperature profile):

LSILT = low shear/low temperature

LSIHT = low shear/high temperature

HSILT = high shear/low temperature

HSIHT = high shear/high temperature

76

APPENDIX III

Raw Data1 for Protein Analysis of the Non-Extruded and Extruded Samples

 

 

Extrusion Conditionsz Protein 1 Protein 2 Protein 3
(%) (%) (%)
Okara
Non-Extruded 24.28 23.66 24.28
Flour
Non-Extruded 8.56 8.74 8.32
FlourNVater Rep 1(HSIHT) 8.66 8.55 -
FlourNVater Rep 2(HSIHT) 8.68 8.19 -
Formulation A
Non-Extruded 9.77 9.77 9.70
LSILT Rep 1 9.68 9.61 -
LSILT Rep 2 9.67 9.53 -
LSIHT Rep 1 9.73 9.80 -
LSIHT Rep 2 9.45 10.04 -
HSILT Rep 1 10.01 9.70 -
HSILT Rep 2 9.76 9.80 -
HSIHT Rep 1 10.10 9.72 -
HSIHT Rep 2 9.54 9.86 -
Formulation B
Non-Extruded 10.50 10.46 10.35
LSILT Rep 1 10.08 10.62 -
LSILT Rep 2 10.26 10.69 -
LSIHT Rep 1 10.09 10.51 -
LSIHT Rep 2 10.70 10.25 -
HSILT Rep 1 10.60 10.44 -
HSILT Rep 2 10.35 10.16 -
HSIHT Rep 1 10.22 10.52 -
HSIHT Rep 2 10.44 10.34 -

 

1All data expressed on a dry basis.
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

77

APPENDIX IV

Raw Data1 for Ash Analysis of the Non-Extruded and Extruded Samples

 

 

Extrusion Conditions2 Ash 1 Ash 2
(%) (%)
Okara
Non-Extruded 4.04 4.07
Flour
Non-Extruded 0.56 0.55
Flour/Water Rep 1(HSIHT) 0.57 0.56
FlourNVater Rep 2(HSIHT) 0.56 0.57
Formulation A
Non-Extruded 0.96 0.96
LSILT Rep 1 0.97 0.96
LSILT Rep 2 0.98 0.97
LSIHT Rep 1 0.97 0.98
LSIHT Rep 2 0.98 0.97
HSILT Rep 1 0.98 0.97
HSILT Rep 2 0.97 0.98
HSIHT Rep 1 0.97 0.98
HSIHT Rep 2 0.97 0.98
Formulation B

Non-Extruded 1 .05 1 .08
LSILT Rep 1 1.10 1.09
LSILT Rep 2 1.10 1.09
LSIHT Rep 1 1.10 1.08
LSIHT Rep 2 1.08 1.09
HSILT Rep 1 1.08 1.08
HSILT Rep 2 1.08 1.08
HSIHT Rep 1 1.06 1.09
HSIHT Rep 2 1.06 1.06

 

1All data expressed on a dry basis.
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

78

APPENDIX V

Raw Data‘ for Pat Analysis of the Non-Extruded and Extruded Samples

 

 

Extrusion Conditionsz Fat 1 Fat 2 Fat 3
(%) (%) (%)
Okara
Non-Extnlded 9.90 9.73 9.87
Flour
Non-Extruded 1 .04 1 .00 1.06
FlourNVater Rep 1(HSIHT) 0.99 0.96 1.03
Flour/Water Rep 2(HSIHT) 0.97 0.96 0.92
Formulation A
Non-Extruded 1 .60 1 .56 1.61
LSILT Rep 1 1.51 1.58 1.58
LSILT Rep 2 1.54 1.61 1.58
LSIHT Rep 1 1.49 1.55 1.58
LSIHT Rep 2 1.54 1.49 1.58
HSILT Rep 1 1.50 1.55 1.58
HSILT Rep 2 1.57 1.50 1.58
HSIHT Rep 1 1.49 1.57 1.55
HSIHT Rep 2 1.55 1.54 1.48
Formulation B

Non-Extruded 1 .80 1 .77 1.84
LSILT Rep 1 1.71 1.73 1.76
LSILT Rep 2 1.75 1.78 1.73
LSIHT Rep 1 1.70 1.77 1.73
LSIHT Rep 2 1.79 1.77 1.69
HSILT Rep 1 1.71 1.77 1.80
HSILT Rep 2 1.76 1.72 1.80
HSIHT Rep 1 1.74 1.78 1.69
HSIHT Rep 2 1.70 1.79 1.77

 

1All data expressed on a dry basis.
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

79

APPENDIX VI

Raw Data1 for Water Absorption Index (WAI) Analysis of the Non-Extruded
and Extruded Samples

 

 

Extrusion Conditions“ WAI 1 WAI 2
Okara
Non-Extruded 4.74 4.74
Flour
Non-Extruded 1 .90 1.91
FlourNVater Rep 1(HSIHT) 7.62 7.48
FlourNVater Rep 2(HSIHT) 7.73 7.71
Formulation A
Non-Extruded 2.08 2.09
LSILT Rep 1 6.55 6.60
LSILT Rep 2 6.52 6.50
LSIHT Rep 1 6.95 6.93
LSIHT Rep 2 6.86 6.86
HSILT Rep 1 6.30 6.65
HSILT Rep 2 7.14 6.72
HSIHT Rep 1 7.60 7.38
HSIHT Rep 2 7.65 7.58
Formulation B
Non-Extruded 2.12 2.14
LSILT Rep 1 5.39 5.43
LSILT Rep 2 5.53 5.55
LSIHT Rep 1 6.35 6.24
LSIHT Rep 2 6.23 6.11
HSILT Rep 1 6.87 6.73
HSILT Rep 2 6.66 6.62
HSIHT Rep 1 6.87 6.71
HSIHT Rep 2 6.62 6.67

 

1All data expressed on a dry basis.
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

80

APPENDIX VII

Raw Data1 for Water Solubility Index (WSI) Analysis of the Non-Extruded
and Extruded Samples

 

 

Extrusion Conditions“ wsr 1 (%) WSI 2 (%)
Okara
Non-Extruded 7.49 7.16
Flour
Non-Extruded 4.80 4.88
Flour/Water Rep 1(HSIHT) 8.35 8.33
Flour/Water Rep 2(HSIHT) 8.35 7.98

Formulation A

Non-Extruded 5.83 5.83
LSILT Rep 1 9.40 9.15
LSILT Rep 2 9.21 9.03
LSIHT Rep 1 10.09 9.75
LSIHT Rep 2 9.36 9.33
HSILT Rep 1 8.21 8.75
HSILT Rep 2 10.39 10.89
HSIHT Rep 1 9.36 9.92
HSIHT Rep 2 10.15 10.21

Formulation B

Non-Extruded 6.12 6.1 1
LSILT Rep 1 6.83 6.97
LSILT Rep 2 7.46 7.57
LSIHT Rep 1 9.13 9.02
LSIHT Rep 2 8.85 9.26
HSILT Rep 1 9.23 8.88
HSILT Rep 2 8.05 7.73
HSIHT Rep 1 10.06 9.59
HSIHT Rep 2 8.25 8.10

 

1All data expressed on a dry basis.
2Extrusion conditions used (screw configuration/temperature profile):

LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

81

APPENDIX VIII

Raw Data for the Radial Expansion Ratio1 Analysis of the Extruded
Samples

 

Extrusion 1 2 3 4 5 6 7 8 9 10 Avg Ratio
Conditions2

 

Flour
HSIHT Rep 1 4.6 4.5 4.6 4.8 4.8 4.7 4.5 4.8 4.8 4.7 4.68 1.56
HSIHT Rep 2 4.7 4.7 4.8 4.5 4.8 4.8 4.9 4.7 4.5 4.6 4.70 1.57
Formulation A
LSILT Rep 1 4.2 4.0 3.8 4.1 3.7 3.8 3.5 3.8 3.5 3.5 3.79 1.26
LSILT Rep 2 3.7 3.9 3.6 3.8 4.1 3.7 3.9 3.6 3.5 3.5 3.73 1.24
LSIHT Rep 1 3.0 3.2 3.4 3.4 3.0 3.5 3.1 3.2 3.2 3.2 3.22 1.07
LSIHT Rep 2 3.3 3.3 3.4 3.0 3.1 3.2 3.4 3.3 3.2 3.0 3.22 1.07
HSILT Rep 1 2.9 3.1 3.0 2.9 3.1 3.2 3.3 3.0 2.8 3.1 3.04 1.01
HSILT Rep 2 3.0 3.0 3.1 2.9 2.8 3.1 3.0 2.9 3.2 2.9 2.99 1.00
HSIHT Rep 1 3.0 2.7 2.8 3.1 3.2 3.2 3.1 2.8 3.0 2.9 2.98 0.99
HSIHT Rep 2 2.9 2.9 3.0 2.8 2.9 3.1 2.7 2.8 2.9 3.0 2.90 0.97

Formulation B
LSILT Rep 1 3.1 2.9 2.8 3.0 3.1 3.2 2.8 2.9 3.0 3.0 2.98 0.99
LSILT Rep 2 3.1 2.9 2.9 3.0 2.9 2.9 3.0 3.0 2.9 2.8 2.94 0.98
LSIHT Rep 1 3.0 3.1 2.7 2.9 3.0 3.0 3.0 2.7 2.8 2.9 2.91 0.97
LSIHT Rep 2 2.6 3.0 2.9 3.1 2.8 3.0 2.9 2.8 2.8 2.7 2.86 0.95
HSILT Rep 1 2.9 3.0 3.0 2.8 2.7 2.7 2.9 2.6 2.9 2.9 2.84 0.95
HSILT Rep 2 2.8 2.6 3.0 2.9 2.9 2.8 2.9 2.6 2.9 2.8 2.82 0.94
HSIHT Rep 1 2.7 2.7 2.8 2.7 3.0 2.9 2.7 2.8 2.8 2.9 2.80 0.93
HSIHT Rep 2 2.6 2.8 2.8 2.8 2.7 2.8 2.8 2.9 2.7 2.8 2.77 0.92

1Radial Expansion Ratio was determined by averaging ten diametrical
measurements (mm) of the extruded samples and then dividing by the diameter
of the die opening (3 mm).
2Extrusion conditions used (screw configuration/temperature profile):

LSILT = low shear/low temperature

LSIHT = low shear/high temperature

HSILT = high shear/low temperature

HSIHT = high shear/high temperature

82

APPENDIX IX

Raw Data for Bulk Density (80) Analysis of the Extruded Samples

 

Extrusion Conditions‘ 80 1 so 2 so 3
(gl100ml) (gl100ml) (gl100ml)

 

Flour
FlourNVater Rep 1(HSIHT) 48.6 48.2 48.3
FlourNVater Rep 2(HSIHT) 49.7 49.3 49.0
Formulation A
LSILT Rep 1 52.5 52.7 53.5
LSILT Rep 2 49.7 49.9 49.4
LSIHT Rep 1 55.4 55.9 55.5
LSIHT Rep 2 55.4 56.0 55.7
HSILT Rep 1 56.8 56.9 57.4
HSILT Rep 2 57.0 57.7 57.2
HSIHT Rep 1 59.5 59.9 59.3
HSIHT Rep 2 58.9 59.3 59.3
Formulation B
LSILT Rep 1 55.6 56.3 55.7
LSILT Rep 2 55.0 55.3 55.5
LSIHT Rep 1 56.2 56.0 55.8
LSIHT Rep 2 58.8 59.0 58.7
HSILT Rep 1 60.9 61.2 60.8
HSILT Rep 2 61.8 61.5 61.4
HSIHT Rep 1 63.8 64.2 63.6
HSIHT Rep 2 62.5 62.3 62.0

 

1Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

83

APPENDIX X

Raw Data for Breaking Strength2 (BS) Analysis of the Extruded Samples

 

 

 

 

Extrusion as 1 as 2 es 3 as 4 as 5 sw’ Avg

Conditions’ W) IN) (N) (N) (NI (9) 33
(N19)

Flour
Flour/Water Rep 1 193.88 203.40 193.84 207.03 225.52 6.90 29.67
F lourNVater Rep 2 234.10 256.89 266.75 250.74 230.45 6.70 36.98
Formulation A
LSILT Rep 1 299.04 261.79 268.05 258.44 230.41 7.20 36.60
LSILT Rep 2 293.98 301.01 274.85 312.36 289.26 7.30 40.31
LSIHT Rep 1 310.65 316.60 267.75 276.43 321.15 7.50 39.80
LSIHT Rep 2 336.43 302.39 297.02 277.92 264.30 7.50 39.42
HSILT Rep 1 343.77 306.07 291.22 354.37 346.01 7.80 42.09
HSILT Rep 2 329.01 352.93 304.42 377.79 352.49 8.00 42.99
HSIHT Rep 1 427.28 397.22 385.79 443.33 381.65 8.50 47.89
HSIHT Rep 2 400.36 343.91 341.59 387.81 371.57 8.70 42.42
Formulation B

LSILT Rep 1 340.79 362.44 350.44 344.47 365.10 8.50 41.49
LSILT Rep 2 291.97 330.01 349.77 344.76 317.97 8.30 38.70
LSIHT Rep 1 406.99 445.44 420.36 426.08 380.87 8.90 46.74
LSIHT Rep 2 378.94 383.89 383.38 341.45 352.49 8.80 41.82
HSILT Rep 1 457.96 399.76 437.96 416.48 394.95 8.90 46.74
HSILT Rep 2 403.84 386.52 388.03 395.54 427.50 8.90 44.98
HSIHT Rep 1 420.08 462.46 402.15 417.25 486.44 9.20 47.57
HSIHT Rep 2 396.50 419.44 416.66 422.53 408.51 8.80 46.90

 

1Breaking strength was determined by averaging five breaking strength values

i

3SW = Sample weight used to determine breaking strength

N) and then dividing by the sample weight (g).
Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature

LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

84

APPENDIX XI

Raw Data for Viscosity Properties1 of the Extruded Samples

 

 

 

 

 

 

 

Extrusion Conditions“ Peak Breakdown Setback Final
Viscosity Viscosity
Flour
Non-Extruded 490 489 320 323 236 235 405 401
FlourNVater Rep 1(HSIHT) 443 451 410 420 45 47 77 78
FlourNVater Rep 2(HSIHT) 523 513 491 483 46 47 77 77
Formulation A
Non-Extruded 434 428 281 271 213 193 360 320
LSILT Rep 1 256 259 218 221 52 52 89 89
LSILT Rep 2 277 288 240 251 54 54 90 91
LSIHT Rep 1 288 294 255 261 44 45 77 79
LSIHT Rep 2 339 343 308 312 46 45 76 76
HSILT Rep 1 305 299 269 263 55 56 91 92
HSILT Rep 2 273 280 239 247 53 52 86 85
HSIHT Rep 1 505 590 472 459 47 48 80 79
HSIHT Rep 2 409 393 376 362 52 53 85 84
Formulation B
Non-Extruded 398 402 271 275 193 191 320 319
LSILT Rep 1 401 394 352 346 70 71 118 119
LSILT Rep 2 343 393 304 293 59 59 98 98
LSIHT Rep 1 292 297 246 255 48 48 85 84
LSIHT Rep 2 276 275 235 233 58 59 98 99
HSILT Rep 1 283 284 245 248 57 59 95 95
HSILT Rep 2 326 322 287 283 58 60 97 98
HSIHT Rep 1 249 244 214 209 51 51 87 86
HSIHT Rep 2 261 264 226 229 50 50 86 85

 

 

1\fIscosity properties were determined using the Rapid VIsco-Analyser and were
expressed in Rapid VIsco Units (RVU); each test was run in duplicate.
2Extrusion conditions used (screw configuration/temperature profile):

LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature

HSIHT = high shear/high temperature

85

APPENDIX XII

Raw Data for Screw Speed, Torque, Feed Rate, and Specific Mechanical
Energy (SME) for the Extruded Samples

 

 

Extrusion ConditionsT Screw Speed Torque Feed Rate SME
(TPMI (%) (kg/hr) (kW NR9)
Flour
FlourNVater Rep 1(HSIHT) 100 30 3.0 0.0399
FlourNVater Rep 2(HSIHT) 100 30 3.0 0.0399
Formulation A
LSILT Rep 1 100 40 1.95 0.0824
LSILT Rep 2 100 45 1.95 0.0927
LSIHT Rep 1 100 35 1.95 0.0721
LSIHT Rep 2 100 40 1.95 0.0824
HSILT Rep 1 100 40 1.95 0.0824
HSILT Rep 2 100 40 1.95 0.0824
HSIHT Rep 1 100 30 1.95 0.0721
HSIHT Rep 2 100 35 1.95 0.0618
Formulation B
LSILT Rep 1 100 30 3.71 0.0324
LSILT Rep 2 100 40 3.71 0.0432
LSIHT Rep1 100 30 3.71 0.0324
LSIHT Rep 2 100 35 3.71 0.0378
HSILT Rep 1 100 35 3.71 0.0324
HSILT Rep 2 100 35 3.71 0.0378
HSIHT Rep 1 100 30 3.71 0.0324
HSIHT Rep 2 100 30 3.71 0.0324

 

1Extrusion conditions used (screw configuration/temperature profile):
LSILT = low Shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

86

APPENDIX XIII

Raw Data for Insoluble, Soluble, and Total Dietary Fiber ‘ Analysis of the
Non-Extruded and Extruded Samples

 

 

 

 

 

 

Extrusion Conditionsz Insoluble Soluble Total
(%) (%) (%)
Okara
Non-Extruded 43.24 43.30 10.06 9.89 53.30 53.19
Flour
Non-Extruded 2.00 2.44 1.75 1.67 3.75 4.1 1
FlourNVater Rep 1(HSIHT) 1.38 1.23 2.11 2.30 3.49 3.53
FlourNVater Rep 2(HSIHT) 1.59 1.31 1.90 2.18 3.49 3.49
Formulation A
Non-Extruded 7.73 7.59 1.86 2.29 9.59 9.88
LSILT Rep 1 6.51 6.70 2.49 2.37 9.00 9.07
LSILT Rep 2 6.31 6.13 3.06 3.37 9.37 9.50
LSIHT Rep 1 6.57 6.99 3.15 2.59 9.72 9.58
LSIHT Rep 2 6.52 6.44 3.41 3.47 9.93 9.91
HSILT Rep 1 5.99 5.89 3.50 3.47 9.49 9.36
HSILT Rep 2 5.60 5.73 3.93 3.87 9.53 9.60
HSIHT Rep 1 5.83 5.88 3.60 3.46 9.43 9.34
HSIHT Rep 2 5.61 5.50 3.78 4.04 9.39 9.54
Formulation B
Non-Extruded 8.66 9.02 1.48 1.43 10.14 10.45
LSILT Rep 1 8.47 8.47 2.33 2.30 10.80 10.77
LSILT Rep 2 8.53 8.50 2.14 2.11 10.67 10.61
LSIHT Rep 1 7.44 7.56 3.11 2.98 10.55 10.54
LSIHT Rep 2 7.13 7.11 3.27 3.37 10.40 10.48
HSILT Rep 1 8.23 8.30 2.95 2.93 11.18 11.23
HSILT Rep 2 8.29 8.39 3.35 3.14 11.64 11.53
HSIHT Rep 1 6.96 6.91 3.44 3.47 10.40 10.38
HSIHT Rep 2 6.48 6.52 3.76 3.90 10.24 10.42

 

;All data expressed on a dry basis; all samples were run in duplicate.
2Extrusion conditions used (screw configuration/temperature profile):

LS/ LT

LSIHT =

low shear/low temperature

low Shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

87

 

APPENDIX XIV

Raw Data for Isoflavone1 Analysis of the Non-Extruded and Extruded
Samples

 

 

 

 

 

 

 

 

 

Extrusion Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6
Conditions’
Okara

Non-Extruded 108 109 142 140 397 402 483 494 81 78 24 25

Formulation A

Non-Extruded 82 84 68 63 358 362 462 469 142 142 104 103
LSILT Rep1 169 162 166 162 294 290 333 330 121 118 36 35
LSILT Rep 2 182 170 193 186 313 308 326 318 123 122 36 36
LSIHT Rep 1 199 206 214 216 172 168 182 175 215 222 43 42
LSIHT Rep 2 190 197 191 184 116 122 122 121 256 257 67 64
HSILT Rep 1 138 131 186 175 265 258 263 264 121 117 36 38
HSILT Rep 2 169 178 208 200 273 246 278 262 121 1 16 33 35
HSIHT Rep 1 258 270 272 268 141 141 167 163 198 202 27 26
HSIHT Rep 2 264 151 336 316 120 124 124 131 223 223 22 21

Formulation B .

Non-Extruded 90 90 42 47 360 361 444 451 146 148 1 15 117
LSILT Rep1 144 133 165 192 302 317 348 347 121 117 48 47
LSILT Rep 2 164 171 200 195 355 371 362 375 90 88 24 23
LSIHT Rep1 178 184 182 179 175 167 215 211 167 168 42 40
LSIHT Rep 2 226 215 243 256 141 136 144 153 191 197 27 28
HSILT Rep 1 191 192 222 218 277 282 316 317 118 114 38 37
HSILT Rep 2 189 194 208 210 284 270 317 303 90 93 26 25
HSIHT Rep1 177 184 217 220 157 161 165 160 190 190 46 45
HSIHT Rep 2 210 220 235 233 118 115 144 148 171 168 32 33

 

 

1All data expressed on a dry basis and units are ug isoflavone/g dry
okara; all samples were run in duplicate. Peaks are (in order of elution):
Peak 1 = Genistin
Peak 2 = Daidzin
Peak 3 = Malonyl Daidzin
Peak 4 = Malonyl Genistin
Peak 5 = Acetyl Genistin
Peak 6 = Genistein
2Extrusion conditions used (screw configuration/temperature profile):
LSILT = low shear/low temperature
LSIHT = low shear/high temperature
HSILT = high shear/low temperature
HSIHT = high shear/high temperature

88

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